Dry powder formulations and methods for treating pulmonary diseases

ABSTRACT

The present invention is directed toward respirable dry particles for delivery of divalent metal cation salts and/or monovalent cation salts to the respiratory tract and methods for treating a subject having a respiratory disease and/or infection.

RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/US2011/049435, filed Aug. 26, 2011, published in English, andclaims the benefit of U.S. Patent Application No. 61/431,242 filed onJan. 10, 2011, the benefit of 61/387,925, filed on Sep. 29, 2010, andthe benefit of U.S. Patent Application No. 61/378,146 filed on Aug. 30,2010, the entire teachings of these applications are incorporated hereinby reference.

GOVERNMENT SUPPORT

This invention was made with government support under W911NF-10-1-0382awarded by the Defense Advanced Research Projects Agency. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Pulmonary delivery of therapeutic agents can offer several advantagesover other modes of delivery. These advantages include rapid onset, theconvenience of patient self-administration, the potential for reduceddrug side-effects, ease of delivery by inhalation, the elimination ofneedles, and the like. Inhalation therapy is capable of providing a drugdelivery system that is easy to use in an inpatient or outpatientsetting, results in very rapid onset of drug action, and producesminimal side effects.

Metered dose inhalers (MDIs) are used to deliver therapeutic agents tothe respiratory tract. MDIs are generally suitable for administeringtherapeutic agents that can be formulated as solid respirable dryparticles in a volatile liquid under pressure. Opening of a valvereleases the suspension at relatively high velocity. The liquid thenvolatilizes, leaving behind a fast-moving aerosol of dry particles thatcontain the therapeutic agent. MDIs are reliable for drug delivery tothe upper and middle airways but are limited because they typicallydeliver only low doses per actuation. However, it is the bronchioles andalveoli that are often the site of manifestation of pulmonary diseasessuch as asthma and infections.

Liquid aerosol delivery is one of the oldest forms of pulmonary drugdelivery. Typically, liquid aerosols are created by an air jetnebulizer, which releases compressed air from a small orifice at highvelocity, resulting in low pressure at the exit region due to theBernoulli effect. See, e.g., U.S. Pat. No. 5,511,726. The low pressureis used to draw the fluid to be aerosolized out of a second tube. Thisfluid breaks into small droplets as it accelerates in the air stream.Disadvantages of this standard nebulizer design include relatively largeprimary liquid aerosol droplet size often requiring impaction of theprimary droplet onto a baffle to generate secondary splash droplets ofrespirable sizes, lack of liquid aerosol droplet size uniformity,significant recirculation of the hulk drug solution, and low densitiesof small respirable liquid aerosol droplets in the inhaled air.

Ultrasonic nebulizers use flat or concave piezoelectric disks submergedbelow a liquid reservoir to resonate the surface of the liquidreservoir, forming a liquid cone which sheds aerosol particles from itssurface (U.S. 2006/0249144 and U.S. Pat. No. 5,551,416). Since noairflow is required in the aerosolization process, high aerosolconcentrations can be achieved, however the piezoelectric components arerelatively expensive to produce and are inefficient at aerosolizingsuspensions, requiring active drug to be dissolved at low concentrationsin water or saline solutions. Newer liquid aerosol technologies involvegenerating smaller and more uniform liquid respirable dry particles bypassing the liquid to be aerosolized through micron-sized holes. See,e.g., U.S. Pat. No. 6,131,570; U.S. Pat. No. 5,724,957; and U.S. Pat.No. 6,098,620. Disadvantages of this technique include relativelyexpensive piezoelectric and fine mesh components as well as fouling ofthe holes from residual salts and from solid suspensions.

Dry powder inhalation has historically relied on lactose blending toallow for the dosing of particles that are small enough to be inhaled,but aren't dispersible enough on their own. This process is known to beinefficient and to not work for some drugs. Several groups have tried toimprove on these shortcomings by developing dry powder inhaler (DPI)formulations that are respirable and dispersible and thus do not requirelactose blending. Dry powder formulations for inhalation therapy aredescribed in U.S. Pat. No. 5,993,805 to Sutton et al.; U.S. Pat. No.69,216,527 to Platz et al.; WO 0000176 to Robinson et al.; WO 9916419 toTarara et al.; WO 0000215 to Bot et al; U.S. Pat. No. 5,855,913 to Haneset al.; and U.S. Pat. Nos. 6,136,295 and 5,874,064 to Edwards et al.

Broad clinical application of dry powder inhalation delivery has beenlimited by difficulties in generating dry powders of appropriateparticle size, particle density, and dispersibility, in keeping the drypowder stored in a dry state, and in developing a convenient, hand-helddevice that effectively disperses the respirable dry particles to beinhaled in air. Another limiting factor for long-term storage of drypowders has been the challenge of maintaining stable physicochemicalproperties with the passage of time. In addition, the particle size ofdry powders for inhalation delivery is inherently limited by the factthat smaller respirable dry particles are harder to disperse in air. Drypowder formulations, while offering advantages over cumbersome liquiddosage forms and propellant-driven formulations, are prone toaggregation and low flowability which considerably diminishdispersibility and the efficiency of dry powder-based inhalationtherapies. For example, interparticular Van der Waals interactions andcapillary condensation effects are known to contribute to aggregation ofdry particles. Hickey, A. et al., “Factors Influencing the Dispersion ofDry Powders as Aerosols”, Pharmaceutical Technology, August, 1994.

To overcome interparticle adhesive forces, Batycky et al. in U.S. Pat.No. 7,182,961 teach production of so called “aerodynamically lightrespirable particles,” which have a volume median geometric diameter(VMGD) of greater than 5 microns (μm) as measured using a laserdiffraction instrument such as HELOS (manufactured by Sympatec,Princeton, N.J.). See Batycky et al., column 7, lines 42-65. Anotherapproach to improve dispersibility of respirable particles of averageparticle size of less than 10 μm, involves the addition of a watersoluble polypeptide or addition of suitable excipients (including aminoacid excipients such as leucine) in an amount of 50% to 99.9% by weightof the total composition. Eljamal et al., U.S. Pat. No. 6,582,729,column 4, lines 12-19 and column 5, line 55 to column 6, line 31.However, this approach reduces the amount of active agent that can bedelivered using a fixed amount of powder. Therefore, an increased amountof dry powder is required to achieve the intended therapeutic results,for example, multiple inhalations and/or frequent administration may berequired. Still other approaches involve the use of devices that applymechanical forces, such as pressure from compressed gasses, to the smallparticles to disrupt interparticular adhesion during or just prior toadministration. See, e.g., U.S. Pat. No. 7,601,336 to Lewis et al., U.S.Pat. No. 6,737,044 to Dickinson et al., U.S. Pat. No. 6,546,928 toAshurst et al., or U.S. Pat. Applications 20090208582 to Johnston et al.

A further limitation that is shared by each of the above methods is thatthe aerosols produced typically include substantial quantities of inertcarriers, solvents, emulsifiers, propellants, and other non-drugmaterial. In general, the large quantities of non-drug material arerequired for effective formation of respirable dry particles smallenough for alveolar delivery (e.g. less than 5 microns and preferablyless than 3 microns). However, these amounts of non-drug material alsoserve to reduce the purity and amount of active drug substance that canbe delivered. Thus, these methods remain substantially incapable ofintroducing large active drug dosages accurately to a patient forsystemic delivery.

Therefore, there remains a need for the formation of small particle sizeaerosols that are highly dispersible. Furthermore, there is a need forcreating powders that are dense in mass and in drug, in order tomaximize the quantity of drug within a given delivery container. Inaddition, methods that produce aerosols comprising greater quantities ofdrug and lesser quantities of non-drug material are needed. Finally, amethod that allows a patient to administer a unit dosage rapidly withone or two, small volume breaths is needed.

SUMMARY OF THE INVENTION

The invention relates to respirable dry powders comprised of dryparticles that contain one or more divalent metal cations, such ascalcium (Ca²⁺), as an active ingredient or inactive ingredient, and todry powders that contain the respirable particles. Preferably, therespirable dry particles are small, dense and highly dispersible, asdescribed in detail herein.

In one aspect, the respirable dry powder comprises respirable dryparticles comprising a divalent metal cation salt, a monovalent metalcation salt, one or more additional therapeutic agents, and optionallyan excipient, wherein the ratio of divalent metal cation to monovalentmetal cation is from about 8:1 (mole:mole) to about 2:1 (mole:mole),about 4:1 (mole:mole) to about 2:1 (mole:mole), or 3.9:1 (mole:mole) toabout 2:1 (mole:mole). As shown herein, respirable dry particles thatcontain calcium ions and sodium ions with these ranges provide superiorefficacy. Accordingly, these types of formulations can provide thetherapeutic benefits of the divalent metal cation, and of the additionaltherapeutic agent. Preferably the divalent metal cation salt is acalcium salt such as calcium lactate, calcium sulfate, calciumcarbonate, calcium citrate and combinations thereof. Preferable themonovalent metal cation salt is a lithium salt, a potassium salt or asodium salt. In some embodiments the monovalent metal cation salt is asodium salt selected from the group consisting of sodium chloride,sodium citrate, sodium lactate, sodium sulfate and combinations thereof.When present, the excipient can be present from about 1% (w/w) to about40% (w/w). Preferred excipients are selected from the group consistingof sugars, polysaccharides, sugar alcohols, amino acids, and anycombination thereof. In particular embodiments, the excipient isselected from leucine, maltodextrin, mannitol and any combinationthereof. The additional therapeutic agent comprises from about 0.01%(w/w) to about 90% (w/w) of the respirable dry particles. Suitableadditional therapeutic agents are described herein, and preferred agentsare independently selected from the group consisting of LABAs,short-acting beta agonists, corticosteroids, LAMAs, antibiotics, DNAse,sodium channel blockers and combinations thereof. The respirable dryparticles have a volume median geometric diameter (VMGD) of about 10microns or less; a dispersibility ratio (¼ bar) of 2.0 or less asmeasured by laser diffraction (RODOS/HELOS system); a Fine ParticleFraction (FPF) of less than 5.6 microns of at least 45%, a Fine ParticleFraction (FPF) of less than 3.4 microns of at least 30%, a mass medianaerodynamic diameter (MMAD) of about 7 microns or less, a tap densitygreater than 0.45 g/cc and/or a heat of solution between about −10kcal/mol and 10 kcal/mol.

In another aspect, the respirable dry powder comprises respirable dryparticles which comprise a calcium salt and a sodium salt, wherein theratio of Ca²⁺ to Na⁺ is from about 8:1 (mole:mole) to about 2:1(mole:mole), 4:1 (mole:mole) to about 2:1 (mole:mole), or 3.9:1(mole:mole) to about 2:1 (mole:mole). As shown herein, respirable dryparticles that contain calcium ions and sodium ions with these rangesprovide superior efficacy in certain disease models. The calcium saltcan be selected from the group consisting of calcium lactate, calciumsulfate, calcium carbonate, calcium citrate and combinations thereof.The sodium salt can be selected from the group consisting of sodiumchloride, sodium citrate, sodium lactate, sodium sulfate andcombinations thereof. If desired, the respirable dry powder of thisaspect can further comprise an excipient, which is preferably 1% (w/w)to 40% (w/w) of the dry powder. Preferred excipients are selected fromthe group consisting of sugars, polysaccharides, sugar alcohols, aminoacids, and any combination thereof. In some embodiments, the excipientis selected from leucine, maltodextrin, mannitol and any combinationthereof. The dry powder of this aspect can further comprise anadditional therapeutic agent, such as LABAs, short-acting beta agonists,corticosteroids, LAMAs, antibiotics, DNAse, sodium channel blockers, andcombinations thereof. The respirable dry particles have a volume mediangeometric diameter (VMGD) of about 10 microns or less; a dispersibilityratio (1/4 bar) of 2.0 or less as measured by laser diffraction(RODOS/HELOS system); a Fine Particle Fraction (FPF) of less than 5.6microns of at least 45%, a Fine Particle Fraction (FPF) of less than 3.4microns of at least 30%, a mass median aerodynamic diameter (MMAD) ofabout 7 microns or less, a tap density greater than 0.45 g/cc and/or aheat of solution between about −10 kcal/mol and 10 kcal/mol. Preferably,the calcium cation is present in at least about 5% by weight of therespirable dry powder.

In another aspect, the respirable dry powder comprises respirable dryparticles which comprise a divalent metal cation salt, one or moretherapeutic agents, and optionally an excipient, wherein the respirabledry particles have a volume median geometric diameter (VMGD) of 10microns or less, a dispersibility ratio (1/4 bar) of 2.0 or less asmeasured by laser diffraction (RODOS/HELOS system), and a tap density ofabout 0.4 g/cc to about 1.2 g/cc. In some embodiments, the respirabledry particles have a tap density of about 0.5 g/cc to about 1.2 g/cc. Insome embodiments, the divalent metal cation salt does not have abiological activity selected from the group consisting of anti-bacterialactivity, anti-viral activity, anti-inflammatory activity andcombinations thereof. Preferred divalent metal cation salts for the drypowders of this aspect are magnesium salts, such as of magnesium lactateand magnesium sulfate. In particular embodiments, the respirable dryparticles comprise a) about 20% (w/w) to about 90% (w/w) magnesium salt,and about 0.01% (w/w) to about 20% (w/w) therapeutic agent; b) about 20%(w/w) to about 80% (w/w) magnesium salt, and about 20% (w/w) to about60% (w/w) therapeutic agent; or c) about 5% (w/w) to about 40% (w/w)magnesium salt, and about 60% (w/w) to about 95% (w/w) therapeuticagent; and wherein all components of the respirable dry particles amountto 100 weight %. Preferably, the respirable dry particles comprise 3%(w/w) or greater magnesium ion. The respirable dry powder may containabout 0.01% (w/w) to about 80% (w/w) excipient. Preferred excipients areselected from the group consisting of sugars, polysaccharides, sugaralcohols, amino acids, and any combination thereof. In some embodiments,the excipient is selected from leucine, maltodextrin, mannitol and anycombination thereof. The dry powder of this aspect can further comprisean additional therapeutic agent, such as LABAs, short-acting betaagonists, corticosteroids, LAMAs, antibiotics, DNAse, sodium channelblockers, and combinations thereof. The respirable dry particles have avolume median geometric diameter (VMGD) of about 10 microns or less; adispersibility ratio (1/4 bar) of 2.0 or less as measured by laserdiffraction (RODOS/HELOS system); a Fine Particle Fraction (FPF) of lessthan 5.6 microns of at least 45%, a Fine Particle Fraction (FPF) of lessthan 3.4 microns of at least 30%, a mass median aerodynamic diameter(MMAD) of about 7 microns or less, a tap density greater than 0.45 g/ccand/or a heat of solution between about −10 kcal/mol and 10 kcal/mol.Preferably, the divalent metal cation is present in at least about 5% byweight of the respirable dry powder.

The invention also relates to a respirable dry powder or dry particle,as described herein, for use in therapy (e.g., treatment, prophylaxis,or diagnosis). The invention also relates to the use of a respirable dryparticle or dry powder, as described herein, for use in treatment,prevention or reducing contagion as described herein, and in themanufacture of a medicament for the treatment, prophylaxis or diagnosisof a respiratory disease and/or infection as described herein.

The invention also relates to a method of reducing inflammationcomprising administering to the respiratory tract of a patient in needthereof an effective amount of a respirable dry powder, as describedherein. The inflammation can be associated with asthma, chronicobstructive pulmonary disorder (COPD) or cystic fibrosis (CF).

The invention also relates to a method of treating a respiratory diseasecomprising administering to the respiratory tract of a patient in needthereof an effective amount of a respirable dry powder, as describedherein.

The invention also relates to methods for treating a respiratorydisease, such as asthma, airway hyperresponsiveness, seasonal allergicallergy, bronchiectasis, chronic bronchitis, emphysema, chronicobstructive pulmonary disease, cystic fibrosis and the like, comprisingadministering to the respiratory tract of a subject in need thereof aneffective amount of the respirable dry particles or dry powder. Theinvention also relates to methods for the treatment or prevention ofacute exacerbations of chronic pulmonary diseases, such as asthma,airway hyperresponsiveness, seasonal allergic allergy, bronchiectasis,chronic bronchitis, emphysema, chronic obstructive pulmonary disease,cystic fibrosis and the like, comprising administering to therespiratory tract of a subject in need thereof an effective amount ofthe respirable dry particles or dry powder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F is a table that shows properties for dry powders preparedfrom feedstock Formulations I, II, III and IV described in Examples 1-3and 14. FIG. 1A includes spray drying parameters used for spray dryingthe powders. FIG. 1B shows the HPLC results for percent calcium ioncontent of the powders, density results including tap and bulkdensities, and Karl Fischer results for percent water content in thepowders. FIG. 1C shows fine particle fraction (FPF) data and percentmass of powders collected using a two-stage (ACI-2) Andersen CascadeImpactor. FIG. 1D shows fine particle fraction (FPF) data and percentmass of powders collected using an eight-stage (ACI-8) Andersen CascadeImpactor. FIG. 1E shows data for mass median aerodynamic diameter (MMAD)and FPF (based on total dose and recovered dose). FIG. 1F shows data forvolume median geometric diameter (DV50), geometric standard deviation(GSD) and FPF less than 5.0 microns (FPF<5.0 μm) as measured by Spraytecinstrument and geometric or volume particle size distribution (which isalso referred to as VMGD, ×50/dg or ×50), GSD and 1/4 bar and 0.5/4 barinformation as measured by HELOS with RODOS attachment instrument.

FIG. 2 is a graph that shows a comparison between the average tap andbulk densities for particles prepared from feedstock Formulations I, IIand III and a placebo.

FIG. 3 is a graph that shows a comparison between the particles(prepared from feedstock Formulations I-III and a placebo) at differentdispersion (regulator) pressures for measured volume median geometricdiameter (×50) using a laser diffraction instrument (HELOS with RODOS).

FIG. 4 is a graph that shows a comparison between the particles preparedfrom feedstock Formulations I (identified as PUR111 (Citrate)), II(identified as PUR112 (Sulfate)) and III (identified as PUR113(Lactate)) and a placebo for average FPF obtained by an ACI-2 and ACI-8.

FIG. 5A-D are electron micrographs of Formulation I (FIG. 5A);Formulation III (FIG. 5B); Formulation II (FIG. 5C); and Formulation IV(FIG. 5D)

FIGS. 6A-6B is a table that shows properties for dry powders prepared byfeedstock Formulations 1-9. Formulation 1 in FIG. 6 corresponds toFormulation III-B in Example 2. Formulation 4 in FIG. 6 corresponds toFormulation I-B in Example 1. Formulation 7 in FIG. 6 corresponds toFormulation II-B in Example 3. Abbreviations in the table heading aredescribed elsewhere in the specification. In FIG. 6, all powders weremade using a Büchi spray dryer.

FIG. 7 is a schematic of the pass-through model.

FIG. 8A is a graph showing the results of the bacterial pass-throughmodel with exposure to dry powders. A calcium sulfate-containing powder(4.5 ug Ca/cm² delivered dose) reduced bacterial movement through sodiumalginate mimetic. FIG. 8B is a graph showing the results of thebacterial pass-through model with exposure to dry powders. The calciumsalt dry powders, prepared from the feedstock formulations A-E, testedcontained 0 ug, 4.3 ug, 6.4 ug or 10 ug of calcium. Calcium sulfate (4.3ug Ca/cm² delivered dose), calcium acetate (10 ug Ca/cm² delivered dose)and calcium lactate (6.4 ug Ca/cm² delivered dose) containing powdersreduced bacterial movement through sodium alginate mimetic.

FIG. 9 is a graph that shows the effect of the respirable dry powders,prepared from feedstock formulations 10-1 to 10-4 in Example 10A, onInfluenza A/WSN/33 (H1N1) infection in a dose-dependent manner.

FIG. 10 is a graph that shows the effect of the respirable dry powdersprepared for Example 10B on Influenza A/Panama/99/2007 (H3N2) infectionin a dose-dependent manner.

FIGS. 11A-D are graphs showing that dry powder formulations comprised ofcalcium salts and sodium chloride reduce the severity of influenza inferrets. FIG. 11A shows the changes in body temperature of ferretstreated with a calcium citrate powder compared to the control animals.FIG. 11B shows the changes in body temperature of ferrets treated with acalcium sulfate powder compared to the control animals. FIG. 11C showsthe changes in body temperature of ferrets treated with a calciumlactate powder compared to the control animals. FIG. 11D shows thechange in body temperature from baseline for each animal using areaunder the curve for the duration of the study (d0-d10). Data depict themean±SEM for each group (p=0.09 for the leucine control and lactategroup by Student t-test).

FIG. 12 is a graph showing dry powder formulations consisting ofdifferent excipients (mannitol, maltodextrin) with calcium lactate andsodium chloride reduced influenza titer at higher concentrations thanthe Formulation III powder alone.

FIGS. 13A-C are graphs showing calcium dry powder formulations vary inefficacy against different viral pathogens. Calu-3 cells exposed to noformulation were used as a control and compared to Calu-3 cells exposedto Formulation I, Formulation II, and Formulation III. The concentrationof virus released by cells exposed to each aerosol formulation wasquantified. Symbols represent the mean and standard deviation ofduplicate wells for each test.

FIG. 14 is a graph showing the emitted dose of Formulation III powder atthree different capsule fill weights (25 mg, 60 mg, 75 mg) at varyinginhalation energies.

FIG. 15 is a graph showing the particle size distribution of calciumlactate (Formulation III) powders emitted from different inhalers,characterized by the volume median diameter (Dv50) and plotted againstthe inhalation energy applied. Consistent values of Dv50 at decreasingenergy values indicate that the powder is well dispersed sinceadditional energy does not result in additional deagglomeration of theemitted powder.

FIG. 16 shows a high resolution XRPD pattern of Formulation I powder.This pattern shows that Formulation I powder consists of a combinationof crystalline sodium chloride and a poorly crystalline or amorphouscalcium citrate and potentially calcium chloride-rich phase.

FIG. 17 shows a comparison of XRPD patterns for Formulation I powderwith crystalline reflections from NaCl.

FIG. 18 shows an overlay of temperature cycling DSC thermogram ofFormulation I. A glass transition temperature of approximately 167° C.was observed via cyclic DSC for the amorphous calcium-rich phase.

FIG. 19 shows a high resolution XRPD pattern of Formulation II powder.This pattern shows that Formulation III powder consists of a combinationof crystalline sodium chloride and a poorly crystalline or amorphouscalcium lactate and potentially calcium chloride-rich phase.

FIG. 20 shows a comparison of XRPD patterns for Formulation II powderwith crystalline reflection from NaCl.

FIG. 21 shows an overlay of temperature cycling DSC thermogram ofFormulation II. A glass transition temperature of approximately 144° C.was observed via cyclic DSC for the amorphous calcium-rich phase.

FIG. 22 shows a high resolution XRPD pattern of Formulation IV powder.

FIG. 23 shows a comparison of XRPD patterns for Formulation IV powderwith crystalline reflection from NaCl.

FIG. 24 shows an overlay of temperature cycling DSC thermogram ofFormulation IV. A glass transition temperature of approximately 134° C.was observed via cyclic DSC for the amorphous calcium-rich phase.

FIG. 25A shows a high resolution XRPD pattern of Formulation II powder.This pattern shows that Formulation II has some degree of crystallinecalcium salt content (calcium sulfate) present, in addition tocrystalline sodium chloride. FIG. 25B shows a comparison of XRPDpatterns for Formulation II powder with crystalline reflection fromNaCl.

FIG. 26 shows an overlay of temperature cycling DSC thermogram ofFormulation II. A glass transition temperature of approximately 159° C.was observed via cyclic DSC for the amorphous calcium-rich phase.

FIGS. 27A-H are RAMAN spectra. FIG. 27A shows RAMAN spectra for sixparticles from the Formulation I sample, and are shown overlaid. FIG.27B shows spectrum 389575-6 is background subtracted and overlaid withthe Raman spectra of calcium citrate tetrahydrate, sodium citrate, andleucine. FIG. 27C shows RAMAN spectra for eight particles from theFormulation II sample, and are shown overlaid. FIG. 27D shows spectrum388369-4 is background subtracted and overlaid with Raman spectra ofcalcium sulfate, calcium sulfate dihydrate, sodium sulfate anhydrous,and leucine. FIG. 27E shows RAMAN spectra for twelve particles from theFormulation III sample, and are shown overlaid. FIG. 27F shows spectra389576-7 and 389576-12 are background subtracted and overlaid with theRaman spectra of calcium lactate pentahydrate, and leucine. FIG. 27Gshows RAMAN spectra for twelve particles from the Formulation IV sample,and are shown overlaid. FIG. 27H, spectrum 389577-9 is backgroundsubtracted and overlaid with the Raman spectra of calcium lactatepentahydrate.

FIG. 28 is a graph showing volume particle size results for FormulationII (calcium sulfate) spray dried powders prepared from pre-mixed andstatic mixed liquid feed stocks with increasing solids concentrations.Particle size distribution broadens (increasing GSD) and median volumeparticle size significantly increases (×50) with increasing solidsconcentration in pre-mixed feed stocks. Particle size distributionremains constant with increasing solids concentration in static mixedfeed stocks, while the median volume particle size increases slightly,as expected with increasing solids concentrations.

FIG. 29 is a graph showing volume particle size distribution results forFormulation II (calcium sulfate) spray dried powders prepared frompre-mixed and static mixed liquid feed stocks with increasing solidsconcentrations. Particle size distribution broadens with increasingsolids concentration in pre-mixed feed stocks and remains narrow withincreasing solids concentration in static mixed feed stocks. Triangles 5g/L, static mixed; squares, 5 g/L, pre-mixed; diamonds, 30 g/L, staticmixed; circles 30 g/L, pre-mixed.

FIG. 30 is a graph showing aerosol characterization results forFormulation II (calcium sulfate) spray dried powders prepared frompre-mixed and static mixed liquid feed stocks with increasing solidsconcentration.

FIG. 31A-B are graphs showing the change in fine particle fraction (FPF)of formulations Formulation I (calcium citrate), Formulation II (calciumsulfate), and Formulation III (calcium lactate) during in-use stabilitytesting at extreme conditions. The graph compares change in FPF (totaldose)<5.6 microns (%) versus time elapsed in the chamber at extremetemperature and humidity conditions (30° C., 75% RH). The values in thelegend indicate the true value at time zero. The plots show fluctuationas a function of change as compared to time zero. FIG. 31B is a graphshowing change in volume particle size of formulations Formulation I(calcium citrate), Formulation II (calcium sulfate) and Formulation III(calcium lactate) during in-use stability testing at extreme conditions.The graph compares change in median volume particle size versus timeelapsed in the chamber at extreme temperature and humidity conditions(30° C., 75% RH). The values in the legend indicate the true value attime zero. The plots show fluctuation as a function of change ascompared to time zero. FIG. 31C,D show similar data for a second set ofspray-dried formulations comprised of a control calcium chloride:sodiumchloride:leucine powder and calcium lactate:sodium chloride powderscontaining 10% (i) lactose, (ii) mannitol) or (iii) maltodextrin asexcipients. FIG. 31C compares changes in FPF (total dose)<5.6 microns(%) versus time elapsed in the chamber for the second set of powders atextreme temperature and humidity conditions (30° C., 75% RH). The valuesin the legend indicate the true value at time zero. The plots showfluctuation as a function of change as compared to time zero. FIG. 31Dis a graph showing changes in volume particle sizes of the second set ofpowders during in-use stability testing at extreme conditions. The graphcompares change in median volume particle size versus time elapsed inthe chamber at extreme temperature and humidity conditions (30° C., 75%RH). The values in the legend indicate the true value at time zero. Theplots show fluctuation as a function of change as compared to time zero.

FIG. 32 is a graph showing powder stability for a range of differentpowders as measured by volume particle size upon exposure to ˜40% RHconditions for up to one week.

FIG. 33 is a graph showing volume particle size upon exposure to ˜40% RHconditions for a range of different powders for up to one week. Thisfigure is identical to FIG. 32, except that chloride was removed toallow for better detail.

FIG. 34 is a graph showing a representative TGA thermogram forFormulation I.

FIG. 35 is a graph showing heats of solution obtained upon dissolutionof Formulations I through III. Formulations I through III resulted insignificantly decreased heats of solution as compared to both rawcalcium chloride dihydratedihydrate and a control calciumchloride:sodium chloride:leucine powder.

FIG. 36 is a graph showing the results of an in vivo pneumonia study.Animals treated with Formulation II (calcium sulfate) exhibited 5-foldlower bacterial titers, animals treated with Formulation I (calciumcitrate) exhibited 10.4-fold lower bacterial titers, and animals treatedwith Formulation III (calcium lactate) exhibited 5.9-fold lowerbacterial titers.

FIG. 37 is a table showing the compositions of exemplary dry powderformulations.

FIGS. 38A-38C are graphs showing the results of an in vivo influenzastudy. The graphs show the efficacy of Formulation III at threedifferent doses (0.1 mg, 0.3 mg and 0.9 mg) on body temperature (FIGS.38A and 38B) and body weight (FIG. 38C) ten days following infection.The data indicate that Formulation III is effective in treating ferretflu in a dose-dependent manner.

FIGS. 39A and 39B are graphs showing the efficacy of Formulation III inan OVA mouse model of allergic asthma. The data show that FormulationIII decreases inflammatory cells (eosinophils) associated with asthma.

FIGS. 40A-40D are graphs showing the efficacy of Formulation III oninflammation induced in a tobacco smoke (TS) model of chronicobstructive pulmonary disease (COPD). The data indicate that FormulationIII significantly decreases inflammatory cells associated with COPD.

FIGS. 41A-41B are graphs showing the dispersibility of Formulations III,IV and V. The emitted dose (FIG. 41A) and volume median geometricdiameter (FIG. 41B) of Formulations III, IV and V are shown as afunction of inhaled energy. The data indicate that Formulation IV andFormulation V behave similarly and disperse slightly better thanFormulation III.

FIG. 42 is a graph showing the results of a study of bacterial pneumoniain a mouse model using Formulations III, IV and V. The data show thatFormulation III inhibits bacterial pneumonia more effectively thanFormulation IV and Formulation V.

FIGS. 43A-43B are graphs showing the efficacy of Formulation III,Formulation IV and Formulation V in a mouse OVA model of allergicasthma. The results show that Formulation III decreases total (FIG. 43A)and eosinophil (FIG. 43B) cell counts more effectively than FormulationIV and Formulation V.

FIG. 44 is a graph showing the dispersibility of Formulations III, VI,VII and VIII. The emitted dose of the formulations is shown as afunction of inhaled energy. The data indicate that all the powders arewell-dispersed, with Formulations III, VI and VIII showing a higherdispersibility than Formulation VII.

FIGS. 45A and 45B are graphs illustrating the solid state properties ofFormulation VII and Formulation VIII. FIG. 45A shows a high resolutionXRPD pattern of both Formulation VII and Formulation VIII whichindicates that the powders consists of crystalline leucine and amorphouscalcium lactate and sodium chloride. FIG. 45B shows the mDSC graphs ofboth formulations which indicate the glass transition temperatures ofthe Formulation VII (91° C.) and Formulation VIII (107° C.).

FIGS. 46A and 46B are graphs showing the results of a study of bacterialpneumonia in a mouse model using dry powders having various molar ionratios of calcium to sodium, but a fixed dose of calcium. The dataindicate that all the dry powders are effective in inhibiting bacterialpneumonia.

FIGS. 47A-47B are graphs showing results of an in vivo influenza studyin ferrets. The graphs show the efficacy of Formulation III andFormulation VI on body temperature (FIG. 47A) and body weight (FIG. 47B)ten days following infection. The data indicate that both FormulationIII and Formulation VI are effective in treating ferret flu.

FIGS. 48A-48B are graphs showing the efficacy of dry powders havingvarious molar ion ratios of calcium to sodium, but a fixed dose ofcalcium in an OVA mouse model of allergic asthma. The data show that allformulations decrease total cell (FIG. 48A) and eosinophils cell (FIG.48B) counts and that dry powders with higher molar ratios of calcium tosodium are more effective.

FIGS. 49A-49D are graphs showing the efficacy of Formulation III andFormulation VII on inflammation induced in a TS model of COPD. The dataindicate that both Formulation III and Formulation VII significantlydecreases inflammatory cells associated with COPD and that a once-dailydose (QD) of Formulation III is as effective as a twice daily dose(BID).

FIG. 50A and FIG. 506B. A significant reduction in KC and MIP2, two keyneutrophil chemokines, was seen when TS Mice were treated Q.D. withFormulations III and VII.

FIG. 51A. A significant reduction in neutrophil inflammation, asrepresented by cell counts, was seen at the lowest dose tested, when TSMice were treated B.I.D. with Formulation VIII.

FIG. 51B. A significant reduction in neutrophil inflammation, asrepresented by cell counts, was seen when OVA sensitized mice weretreated with Formation VIII and then infected with rhinovirus.

FIG. 52. No significant increase in airway resistance was observed whenmice were treated with Formulation VIII and then challenged withmethacholine chloride (MCh) as compared to when the sham treatment groupwas challenged with MCh.

FIG. 53 is a graph showing a decrease in airway resistance was observedwhen mice were treated with Formulation XI and 48-A and then challengedwith methacholine chloride (MCh) as compared to when the sham(Placebo-B) treatment group was challenged with MCh.

FIG. 54 is a graph showing a decrease in airway resistance was observedwhen mice were treated with Formulation XIV and 48-B and then challengedwith methacholine chloride (MCh) as compared to when the sham(Placebo-B) treatment group was challenged with MCh.

FIG. 55A is a schematic showing mice sensitized and challenged to OVA.

FIG. 55B is a schematic showing a 4-day TS exposure model.

FIG. 55C is a schematic showing the rhinovirus infection model.

FIG. 55D is a schematic showing the experimental procedure used inExample 45.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates, in part, to respirable dry powders that deliverone or more divalent metal cations, such as calcium, as an activeingredient, and to divalent metal cation-containing (e.g.,calcium-containing) respirable dry particles contained within thepowders. The invention also relates to respirable dry particles thatcontain one or more monovalent cations (such as Na⁺) and to dry powdersthat contain the respirable particles.

In one aspect, the respirable dry powders and dry particles of theinvention may be divalent metal cation (e.g., calcium) dense respirableparticles that are small and dispersible. For example, the dry particlescan contain a high percentage of divalent metal cation salt (i.e., bedense in divalent metal cation salt) and/or contain divalent metalcation salts that dissociate to release two or more moles of divalentmetal cation per mole of salt.

The respirable dry powders and dry particles may contain a highpercentage of a divalent metal cation salt (e.g. a calcium salt) thatdissociates to release one mole of divalent metal cation per mole ofsalt or that contains a high molecular weight anion and thereforedissociates to produce a relatively small mass of divalent cation.Accordingly, in some aspects, the respirable dry powders and dryparticles of the invention may be divalent metal cation salt (e.g.,calcium salt) dense and are small and dispersible.

In another aspect, the respirable dry powders and dry particles are massdense (e.g. have a tap density or envelope density of greater than about0.4 g/cc, or at least about 0.45 g/cc, 0.5 g/cc, 0.6 g/cc, 0.7 g/cc or0.8 g/cc), small, and dispersible.

The respirable dry particles may be large or small, e.g., the dry powderhas a geometric diameter (VMGD) between 0.5 microns and 30 microns.Optionally, the MMAD of the dry powder may be between 0.5 and 10microns, more preferably between 1 and 5 microns. When they are small,the particles optionally have a tap density between 0.4 g/cc and 1.2g/cc, or between 0.55 g/cc and 1.0 g/cc. When they are large, theparticles can have a geometric diameter (VMGD) between 5 microns and 30microns (more preferably between 10 microns and 30 microns), andoptionally, have a tap density between 0.01 g/cc and 0.4 g/cc, orbetween 0.05 g/cc and 0.25 g/cc.

Respirable dry powders that contain small particles and that aredispersible in air, and preferably dense (e.g., dense in activeingredient) are a departure from the conventional wisdom. It is wellknown that the propensity for particles to aggregate or agglomerateincreases as particle size decreases. See, e.g., Hickey, A. et al.,“Factors Influencing the Dispersion of Dry Powders as Aerosols”,Pharmaceutical Technology, August, 1994.

As described herein, the invention provides respirable dry powders thatcontain respirable particles that are small and dispersible in airwithout additional energy sources beyond the subject's inhalation. Thus,the respirable dry powders and respirable dry particles can be usedtherapeutically, without including large amounts of non-activecomponents (e.g., excipients) in the particles or powders, or by usingdevices that apply mechanical forces to disrupt aggregated oragglomerated particles during or just prior to administration. Forexample, devices such as passive dry powder inhalers may be used todeliver the dry powder or dry particles.

The respirable dry powders and respirable particles of the invention arealso generally, dense in active ingredient(s), i.e., divalent metalcations (e.g., calcium containing salt(s)). For example, as describedherein, when an excipient is included in the respirable dry powder orparticles, the excipient is a minor component (e.g., about 50% or less,by weight, preferably about 20% or less by weight, about 12% or less byweight, about 10% or less by weight, about 8% or less by weight or lessby weight). However, in some embodiments, an excipient can be present inhigher amounts. Thus, in one aspect, the respirable particles are notonly small and highly dispersible, but can contain a large amount ofdivalent metal cation, for example, calcium (Ca²⁺). Accordingly, asmaller amount of powder will need to be administered in order todeliver the desired dose of divalent metal cation (e.g., calcium). Forexample, the desired dose of calcium may be delivered with one or twoinhalations from a capsule-type or blister-type inhaler.

Respirable dry powder and dry particles that are small, dispersible anddense (e.g., divalent cation dense, divalent cation salt dense, and/ormass dense) provide advantages for therapeutic uses. For example, adesired therapeutically effective dose of divalent metal cation (e.g.calcium) can be delivered when a subject inhales a small volume of drypowder.

DEFINITIONS

The term “dry powder” as used herein refers to a composition thatcontains finely dispersed respirable dry particles that are capable ofbeing dispersed in an inhalation device and subsequently inhaled by asubject. Such dry powder or dry particle may contain up to about 25%, upto about 20% or up to about 15% water or other solvent, or besubstantially free of water or other solvent, or be anhydrous.

The term “dry particles” as used herein refers to respirable particlesthat may contain up to about 25%, up to about 20%, or up to about 15%water or other solvent, or be substantially free of water or othersolvent, or be anhydrous.

The term “respirable” as used herein refers to dry particles or drypowders that are suitable for delivery to the respiratory tract (e.g.,pulmonary delivery) in a subject by inhalation. Respirable dry powdersor dry particles have a mass median aerodynamic diameter (MMAD) of lessthan about 10 microns, preferably about 5 microns or less.

The term “small” as used herein to describe respirable dry particlesrefers to particles that have a volume median geometric diameter (VMGD)of about 10 microns or less, preferably about 5 microns or less.

As used herein, the terms “administration” or “administering” ofrespirable dry particles refers to introducing respirable dry particlesto the respiratory tract of a subject.

As used herein, the term “respiratory tract” includes the upperrespiratory tract (e.g., nasal passages, nasal cavity, throat, pharynx),respiratory airways (e.g., larynx, tranchea, bronchi, bronchioles) andlungs (e.g., respiratory bronchioles, alveolar ducts, alveolar sacs,alveoli).

The term “dispersible” is a term of art that describes thecharacteristic of a dry powder or dry particles to be dispelled into arespirable aerosol. Dispersibility of a dry powder or dry particles isexpressed herein as the quotient of the volume median geometric diameter(VMGD) measured at a dispersion (i.e., regulator) pressure of 1 bardivided by the VMGD measured at a dispersion (i.e., regulator) pressureof 4 bar, or VMGD at 0.5 bar divided by the VMGD at 4 bar as measured byHELOS/RODOS. These quotients are referred to herein as “1/4 bar,” and“0.5/4 bar,” respectively, and dispersibility correlates with a lowquotient. For example, 1/4 bar refers to the VMGD of respirable dryparticles or powders emitted from the orifice of a RODOS dry powderdisperser (or equivalent technique) at about 1 bar, as measured by aHELOS or other laser diffraction system, divided the VMGD of the samerespirable dry particles or powders measured at 4 bar by HELOS/RODOS.Thus, a highly dispersible dry powder or dry particles will have a 1/4bar or 0.5/4 bar ratio that is close to 1.0. Highly dispersible powdershave a low tendency to agglomerate, aggregate or clump together and/or,if agglomerated, aggregated or clumped together, are easily dispersed ordc-agglomerated as they emit from an inhaler and are breathed in by thesubject. Dispersibility can also be assessed by measuring the sizeemitted from an inhaler as a function of flowrate. As the flow ratethrough the inhaler decreases, the amount of energy in the airflowavailable to be transferred to the powder to disperse it decreases. Ahighly dispersible powder will have its size distribution ascharacterized aerodynamically by its mass median aerodynamic diameter(MMAD) or geometrically by its VMGD, not substantially increase over arange of flow rates typical of inhalation by humans, such as about 15 to60 LPM.

The terms “FPF (<5.6),” “FPF (<5.6 microns),” and “fine particlefraction of less than 5.6 microns” as used herein, refer to the fractionof a sample of dry particles that have an aerodynamic diameter of lessthan 5.6 microns. For example, FPF (<5.6) can be determined by dividingthe mass of respirable dry particles deposited on the stage one and onthe collection filter of a two-stage collapsed Andersen Cascade Impactor(ACI) by the mass of respirable dry particles weighed into a capsule fordelivery to the instrument. This parameter may also be identified as“FPF_TD (<5.6),” where TD means total dose. A similar measurement can beconducted using an eight-stage ACI. The eight-stage ACI cutoffs aredifferent at the standard 60 L/min flowrate, but the FPF_TD (<5.6) canbe extrapolated from the eight-stage complete data set. The eight-stageACI result can also be calculated by the USP method of using the dosecollected in the ACI instead of what was in the capsule to determineFPF.

The terms “FPF (<3.4),” “FPF (<3.4 microns),” and “fine particlefraction of less than 3.4 microns” as used herein, refer to the fractionof a mass of respirable dry particles that have an aerodynamic diameterof less than 3.4 microns. For example, FPF (<3.4) can be determined bydividing the mass of respirable dry particles deposited on thecollection filter of a two-stage collapsed ACI by the total mass ofrespirable dry particles weighed into a capsule for delivery to theinstrument. This parameter may also be identified as “FPF_TD (<3.4),”where TD means total dose. A similar measurement can be conducted usingan eight-stage ACI. The eight-stage ACI result can also be calculated bythe USP method of using the dose collected in the ACI instead of whatwas in the capsule to determine FPF.

The terms “FPF (<5.0),” “FPF (<5.0 microns),” and “fine particlefraction of less than 5.0 microns” as used herein, refer to the fractionof a mass of respirable dry particles that have an aerodynamic diameterof less than 5.0 microns. For example, FPF (<5.0) can be determined byusing an eight-stage ACI at the standard 60 L/min flowrate byextrapolating from the eight-stage complete data set. This parameter mayalso be identified as “FPF_TD (<5.0),” where TD means total dose. Whenused in conjunction with a geometric size distribution such as thosegiven by a Malvern Spraytec, Malvern Mastersizer or Sympatec Helosparticle sizer, “FPF (<5.0)” refers to the fraction of a mass ofrespirable dry particles that have a geometric diameter of less than 5.0micrometers.

The terms “FPD (<4.4)”, “FPD<4.4 μm”, “FPD (<4.4 microns)” and “fineparticle dose of less than 4.4 microns” as used herein, refer to themass of respirable dry powder particles that have an aerodynamicdiameter of less than 4.4 micrometers. For example, FPD<4.4 μm can bedetermined by using an eight-stage ACI at the standard 60 L/min flowrateand summing the mass deposited on the filter, and stages 6, 5, 4, 3, and2 for a single dose of powder actuated into the ACI.

As used herein, the term “emitted dose” or “ED” refers to an indicationof the delivery of a drug formulation from a suitable inhaler deviceafter a firing or dispersion event. More specifically, for dry powderformulations, the ED is a measure of the percentage of powder that isdrawn out of a unit dose package and that exits the mouthpiece of aninhaler device. The ED is defined as the ratio of the dose delivered byan inhaler device to the nominal dose (i.e., the mass of powder per unitdose placed into a suitable inhaler device prior to firing). The ED isan experimentally-measured parameter, and can be determined using themethod of USP Section 601 Aerosols, Metered-Dose Inhalers and Dry PowderInhalers, Delivered-Dose Uniformity, Sampling the Delivered Dose fromDry Powder Inhalers, United States Pharmacopia convention, Rockville,Md., 13^(th) Revision, 222-225, 2007. This method utilizes an in vitrodevice set up to mimic patient dosing.

The term “capsule emitted powder mass” or “CEPM” as used herein, refersto the amount of dry powder formulation emitted from a capsule or doseunit container during an inhalation maneuver. CEPM is measuredgravimetrically, typically by weighing a capsule before and after theinhalation maneuver to determine the mass of powder formulation removed.CEPM can be expressed either as the mass of powder removed, inmilligrams, or as a percentage of the initial filled powder mass in thecapsule prior to the inhalation maneuver.

The term “effective amount,” as used herein, refers to the amount ofagent needed to achieve the desired effect, such as an amount that issufficient to increase surface and/or bulk viscoelasticy of therespiratory tract mucus (e.g., airway lining fluid), increase gelationof the respiratory tract mucus (e.g., at the surface and/or bulkgelation), increase surface tension of the respiratory tract mucus,increasing elasticity of the respiratory tract mucus (e.g., surfaceelasticity and/or bulk elasticity), increase surface viscosity of therespiratory tract mucus (e.g., surface viscosity and/or bulk viscosity),reduce the amount of exhaled particles, reduce pathogen (e.g., bacteria,virus) uptake or pathogen burden, reduce symptoms (e.g., fever,coughing, sneezing, nasal discharge, diarrhea and the like), reduceoccurrence of infection, reduce viral replication, or improve or preventdeterioration of respiratory function (e.g., improve forced expiratoryvolume in 1 second FEV1 and/or forced expiratory volume in 1 second FEV1as a proportion of forced vital capacity FEV1/FVC) or stimulate innateimmunity of airway epithelium. The actual effective amount for aparticular use can vary according to the particular dry powder or dryparticle, the mode of administration, and the age, weight, generalhealth of the subject, and severity of the symptoms or condition beingtreated. Suitable amounts of dry powders and dry particles to beadministered, and dosage schedules, for a particular patient can bedetermined by a clinician of ordinary skill based on these and otherconsiderations.

The term “pharmaceutically acceptable excipient” as used herein meansthat the excipient can be taken into the lungs with no significantadverse toxicological effects on the lungs. Such excipients aregenerally regarded as safe (GRAS) by the U.S. Food and DrugAdministration.

All references to salts herein include anhydrous forms and all hydratedforms of the salt.

Dry Powders and Dry Particles

The invention relates to respirable dry powders and dry particles thatcontain one or more divalent metal cations, such as beryllium (Be²⁺),magnesium, (Mg²⁺), calcium (Ca²⁺), strontium (Sr²⁺), barium (Ba²⁺),radium (Ra²⁺), or iron (ferrous ion, Fe²⁺), as an active ingredient. Theactive divalent metal cation (e.g., calcium) is generally present in thedry powders and dry particles in the form of a salt, which can becrystalline or amorphous. The dry powders and dry particles canoptionally include additional salts (e.g. monovalent salts, such assodium salts, potassium salts, and lithium salts.), therapeuticallyactive agents or pharmaceutically acceptable excipients.

In some aspects, the respirable dry powder and dry particles contain oneor more salts of a group IIA element (i.e., one or more beryllium salts,magnesium salts, calcium salts, barium salts, radium salts or anycombination of the forgoing). In more particular aspects, the respirabledry powder and dry particles contain one or more calcium salts,magnesium salts or any combination of the forgoing. In particularembodiments, the respirable dry powder and dry particles contain one ormore calcium salts. In other particular embodiments, respirable drypowder and dry particles contain one or more magnesium salts.

Suitable beryllium salts include, for example, beryllium phosphate,beryllium acetate, beryllium tartrate, beryllium citrate, berylliumgluconate, beryllium maleate, beryllium succinate, sodium berylliummalate, beryllium alpha brom camphor sulfonate, berylliumacetylacetonate, beryllium formate or any combination thereof.

Suitable magnesium salts include, for example, magnesium fluoride,magnesium chloride, magnesium bromide, magnesium iodide, magnesiumphosphate, magnesium sulfate, magnesium sulfite, magnesium carbonate,magnesium oxide, magnesium nitrate, magnesium borate, magnesium acetate,magnesium citrate, magnesium gluconate, magnesium maleate, magnesiumsuccinate, magnesium malate, magnesium taurate, magnesium orotate,magnesium glycinate, magnesium naphthenate, magnesium acetylacetonate,magnesium formate, magnesium hydroxide, magnesium stearate, magnesiumhexafluorsilicate, magnesium salicylate or any combination thereof.

Suitable calcium salts include, for example, calcium chloride, calciumsulfate, calcium lactate, calcium citrate, calcium carbonate, calciumacetate, calcium phosphate, calcium alginate, calcium stearate, calciumsorbate, calcium gluconate and the like.

Suitable strontium salts include, for example, strontium chloride,strontium phosphate, strontium sulfate, strontium carbonate, strontiumoxide, strontium nitrate, strontium acetate, strontium tartrate,strontium citrate, strontium gluconate, strontium maleate, strontiumsuccinate, strontium malate, strontium aspartate in either L and/orD-form, strontium fumarate, strontium glutamate in either L- and/orD-form, strontium glutarate, strontium lactate, strontium L-threonate,strontium malonate, strontium ranelate (organic metal chelate),strontium ascorbate, strontium butyrate, strontium clodronate, strontiumibandronate, strontium salicylate, strontium acetyl salicylate or anycombination thereof.

Suitable barium salts include, for example, barium hydroxide, bariumfluoride, barium chloride, barium bromide, barium iodide, bariumsulfate, barium sulfide (S), barium carbonate, barium peroxide, bariumoxide, barium nitrate, barium acetate, barium tartrate, barium citrate,barium gluconate, barium maleate, barium succinate, barium malate,barium glutamate, barium oxalate, barium malonate, barium naphthenate,barium acetylacetonate, barium formate, barium benzoate, bariump-t-butylbenzoate, barium adipate, barium pimelate, barium suberate,barium azelate, barium sebacate, barium phthalate, barium isophthalate,barium terephthalate, barium anthranilate, barium mandelate, bariumsalicylate, barium titanate or any combination thereof.

Suitable radium salts include, for example, radium fluoride, radiumchloride, radium bromide, radium iodide, radium oxide, radium nitride orany combination thereof.

Suitable iron (ferrous) salts include, for example, ferrous sulfate,ferrous oxides, ferrous acetate, ferrous citrate, ferrous ammoniumcitrate, ferrous ferrous gluconate, ferrous oxalate, ferrous fumarate,ferrous maleate, ferrous malate, ferrous lactate, ferrous ascorbate,ferrous erythrobate, ferrous glycerate, ferrous pyruvate or anycombination thereof.

In one aspect, the dry particles of the invention are small, andpreferably divalent metal cation (e.g., calcium) dense, and aredispersible. In another aspect of the invention, the dry particles aresmall, dense in divalent metal cation salt (e.g. contain at least about30% or at least about 40% (w/w) divalent metal cation salt), and aredispersible. In a further aspect of the invention, the dry particles aresmall, dense in mass (e.g. tap density, envelope density), and aredispersible. In this last aspect, the particles can be dense in divalentmetal cation salt (e.g. calcium, magnesium), or can have low loading ofmetal cation salt in the formulation.

Generally, the dry particles of the invention have a VMGD as measured byHELOS/RODOS at 1.0 bar of about 10 μm or less (e.g., about 0.1 μm toabout 10 μm). Preferably, the dry particles of the invention have anVMGD of about 9 μm or less (e.g., about 0.1 μm to about 9 μm), about 8μm or less (e.g., about 0.1 μm to about 8 μm), about 7 μm or less (e.g.,about 0.1 μm to about 7 μm), about 6 μm or less (e.g., about 0.1 μm toabout 6 μm), about 5 μm or less (e.g., less than 5 μm, about 0.1 μm toabout 5 μm), about 4 μm or less (e.g., 0.1 μm to about 4 μm), about 3 μmor less (e.g., 0.1 μm to about 3 μm), about 2 μm or less (e.g., 0.1 μmto about 2 μm), about 1 μm or less (e.g., 0.1 μm to about 1 μm), about 1μm to about 6 μm, about 1 μm to about 5 μm, about 1 μm to about 4 μm,about 1 μm to about 3 μm, or about 1 μm to about 2 μm as measured byHELOS/RODOS at 1.0 bar.

In another aspect, the dry particles of the invention are large, andpreferably calcium dense, and are dispersible. In another aspect of theinvention, the respirable particles are large, dispersible, and have arelatively low loading of the divalent cation and divalent cation salt,e.g. divalent cation salt is 50% or less (w/w). Generally, the dryparticles of the invention have a VMGD as measured by HELOS/RODOS at 1.0bar of about 30 μm or less (e.g., about 5 μm to about 30 μm).Preferably, the dry particles of the invention have an VMGD of about 25μm or less (e.g., about 5 μm to about 25 μm), about 20 μm or less (e.g.,about 5 μm to about 20 μm), about 15 μm or less (e.g., about 5 μm toabout 15 μm), about 12 μm or less (e.g., about 5 μm to about 12 μm),about 10 μm or less (e.g., about 5 μm to about 10 μm), or about 8 μm orless (e.g., 6 μm to about 8 μm) as measured by HELOS/RODOS at 1.0 bar.

In addition, whether the particles are small or large, the dry particlesof the invention are dispersible, and have 1/4 bar and/or 0.5/4 bar ofabout 2.2 or less (e.g., about 1.0 to about 2.2) or about 2.0 or less(e.g., about 1.0 to about 2.0). Preferably, the dry particles of theinvention have 1/4 bar and/or 0.5/4 bar of about 1.9 or less (e.g.,about 1.0 to about 1.9), about 1.8 or less (e.g., about 1.0 to about1.8), about 1.7 or less (e.g., about 1.0 to about 1.7), about 1.6 orless (e.g., about 1.0 to about 1.6), about 1.5 or less (e.g., about 1.0to about 1.5), about 1.4 or less (e.g., about 1.0 to about 1.4), about1.3 or less (e.g., less than 1.3, about 1.0 to about 1.3), about 1.2 orless (e.g., 1.0 to about 1.2), about 1.1 or less (e.g., 1.0 to about 1.1μm) or the dry particles of the invention have 1/4 bar of about 1.0.

Alternatively or in addition, the respirable dry particles of theinvention can have an MMAD of about 10 microns or less, such as an MMADof about 0.5 micron to about 10 microns. Preferably, the dry particlesof the invention have an MMAD of about 5 microns or less (e.g. about 0.5micron to about 5 microns, preferably about 1 micron to about 5microns), about 4 microns or less (e.g., about 1 micron to about 4microns), about 3.8 microns or less (e.g. about 1 micron to about 3.8microns), about 3.5 microns or less (e.g. about 1 micron to about 3.5microns), about 3.2 microns or less (e.g. about 1 micron to about 3.2microns), about 3 microns or less (e.g. about 1 micron to about 3.0microns), about 2.8 microns or less (e.g. about 1 micron to about 2.8microns), about 2.2 microns or less (e.g. about 1 micron to about 2.2microns), about 2.0 microns or less (e.g. about 1 micron to about 2.0microns) or about 1.8 microns or less (e.g. about 1 micron to about 1.8microns).

Alternatively or in addition, the respirable dry powders and dryparticles of the invention can have an FPF of less than about 5.6microns (FPF<5.6 μm) of at least about 20%, at least about 30%, at leastabout 40%, preferably at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, or at least about70%.

Alternatively or in addition, the dry powders and dry particles of theinvention have a FPF of less than 5.0 microns (FPF_TD<5.0 pin) of atleast about 20%, at least about 30%, at least about 45%, preferably atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 65% or at least about 70%. Alternatively or inaddition, the dry powders and dry particles of the invention have a FPFof less than 5.0 microns of the emitted dose (FPF_ED<5.0 μm) of at leastabout 45%, preferably at least about 50%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,or at least about 85%. Alternatively or in addition, the dry powders anddry particles of the invention can have an FPF of less than about 3.4microns (FPF<3.4 μm) of at least about 20%, preferably at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, or at least about 55%.

Alternatively or in addition, the respirable dry powders and dryparticles of the invention have a tap density of about 0.1 g/cm³ toabout 1.0 g/cm³. For example, the small and dispersible dry particleshave a tap density of about 0.1 g/cm³ to about 0.9 g/cm³, about 0.2g/cm³ to about 0.9 g/cm³, about 0.2 g/cm³ to about 0.9 g/cm³, about 0.3g/cm³ to about 0.9 g/cm³, about 0.4 g/cm³ to about 0.9 g/cm³, about 0.5g/cm³ to about 0.9 g/cm³, or about 0.5 g/cm³ to about 0.8 g/cm³, greaterthan about 0.4 g/cc, greater than about 0.5 g/cc, greater than about 0.6g/cc, greater than about 0.7 g/cc, about 0.1 g/cm³ to about 0.8 g/cm³,about 0.1 g/cm³ to about 0.7 g/cm³, about 0.1 g/cm³ to about 0.6 g/cm³,about 0.1 g/cm³ to about 0.5 g/cm³, about 0.1 g/cm³ to about 0.4 g/cm³,about 0.1 g/cm³ to about 0.3 g/cm³, less than 0.3 g/cm³. In a preferredembodiment, tap density is greater than about 0.4 g/cc. In anotherpreferred embodiment, tap density is greater than about 0.5 g/cc.Alternatively, tap density is less than about 0.4 g/cc.

Alternatively or in addition, the respirable dry powders and dryparticles of the invention can have a water or solvent content of lessthan about 25%, less than about 20%, less than about 15% by weight ofthe respirable dry particle. For example, the respirable dry particlesof the invention can have a water or solvent content of less than about25%, less than about 20%, less than about 15% by weight, less than about13% by weight, less than about 11.5% by weight, less than about 10% byweight, less than about 9% by weight, less than about 8% by weight, lessthan about 7% by weight, less than about 6% by weight, less than about5% by weight, less than about 4% by weight, less than about 3% byweight, less than about 2% by weight, less than about 1% by weight or beanhydrous. The respirable dry particles of the invention can have awater or solvent content of less than about 6% and greater than about1%, less than about 5.5% and greater than about 1.5%, less than about 5%and greater than about 2%, about 2%, about 2.5%, about 3%, about 3.5%,about 4%, about 4.5%, or about 5%.

As described herein, some respirable dry particles of the inventioncontain one or more divalent metal cations (e.g., calcium (Ca²⁺)) as anactive ingredient which is generally present in the form of a salt(e.g., crystalline and/or amorphous). Suitable calcium salts that can bepresent in the respirable dry particles of the invention include, forexample, calcium chloride, calcium sulfate, calcium lactate, calciumcitrate, calcium carbonate, calcium acetate, calcium phosphate, calciumalginate, calcium stearate, calcium sorbate, calcium gluconate and thelike. In certain preferred aspects, the dry powder or dry particles ofthe invention do not contain calcium phosphate, calcium carbonate,calcium alginate, calcium sterate or calcium gluconate. In anotherpreferred aspect, the dry powder or dry particles of the inventioninclude calcium citrate, calcium lactate, calcium chloride, calciumsulfate, or any combination of these salts. In another preferred aspect,the dry powder or dry particles include calcium citrate, calciumlactate, or any combination of these salts. In another preferred aspect,the dry powder or dry particles include calcium carbonate. In a furtheraspect, the dry powder or dry particles include calcium citrate, calciumlactate, calcium sulfate, calcium carbonate, or any combination of thesesalts. A preferred calcium salt is calcium lactate. In a certainpreferred aspect, the dry powder or dry particles of the invention donot contain calcium chloride. If desired, the respirable dry particlesof the invention contain a divalent metal cation salt (e.g., a calciumsalt) and further contain one or more additional salts, such as one ormore non-toxic salts of the elements sodium, potassium, magnesium,calcium, aluminum, silicon, scandium, titanium, vanadium, chromium,cobalt, nickel, copper, manganese, zinc, tin, silver and the like.Preferably, the dry particles contain at least one calcium salt and atleast one monovalent cation salt (e.g., a sodium salt).

Suitable sodium salts that can be present in the respirable dryparticles of the invention include, for example, sodium chloride, sodiumcitrate, sodium sulfate, sodium lactate, sodium acetate, sodiumbicarbonate, sodium carbonate, sodium stearate, sodium ascorbate, sodiumbenzoate, sodium biphosphate, sodium phosphate, sodium bisulfite, sodiumborate, sodium gluconate, sodium metasilicate and the like. In apreferred aspect, the dry powders and dry particles include sodiumchloride, sodium citrate, sodium lactate, sodium sulfate, or anycombination of these salts.

Suitable lithium salts include, for example, lithium chloride, lithiumbromide, lithium carbonate, lithium nitrate, lithium sulfate, lithiumacetate, lithium lactate, lithium citrate, lithium aspartate, lithiumgluconate, lithium malate, lithium ascorbate, lithium orotate, lithiumsuccinate or and combination thereof.

Suitable potassium salts include, for example, potassium chloride,potassium bromide, potassium iodide, potassium bicarbonate, potassiumnitrite, potassium persulfate, potassium sulfite, potassium bisulfite,potassium phosphate, potassium acetate, potassium citrate, potassiumglutamate, dipotassium guanylate, potassium gluconate, potassium malate,potassium ascorbate, potassium sorbate, potassium succinate, potassiumsodium tartrate and any combination thereof.

In another aspect of the invention, the respirable dry powders orrespirable dry particles are suitable for use as carrier particles fordelivering a therapeutic agent. In these aspects, the respirable drypowders contain respirable dry particles that contain one or moredivalent metal cations that 1) does not on its own have apharmacological effect (e.g., magnesium (Mg²⁺), 2) or is present in anamount that does not produce therapeutic efficacy (e.g., asub-therapeutic amount such as a low % of divalent metal cation salt(e.g., less than about 20%, 15%, 10%, 5% or 3% (w/w)). Preferably, thepharmacological effect is a biological activity selected fromanti-bacterial activity, anti-viral activity, anti-inflammatory activityand combinations thereof. Whether a divalent metal cation, on its own,has such a pharmacological effect can be easily evaluated using the invivo models disclosed and exemplified herein. For example, as usedherein, a divalent metal cation does not have anti-bacterial activitywhen it results in less than 50% reduction in colony forming unitsrecovered from the lung in the mouse model of bacterial pneumoniadisclosed in Example 26. As used herein, a divalent metal cation doesnot have anti-viral activity when it results in less than 50% reductionin nasal wash viral titer in the ferret model of influenza infectiondisclosed in Example 11. As used herein, a divalent metal cation doesnot have anti-inflammatory activity when it results in less than 15%reduction in neutrophils recovered from the lung in the tobacco smokemouse model of COPD disclosed in Example 30. The models and tests arerun substantially as described herein, but substituting the divalentmetal cation to be tested for the formulation in the examples. Thesemodels can also be used to assess therapeutic efficacy of divalent metalcations, such as calcium cations. For example, low calcium loading in adry powder may not produce therapeutic efficacy because the quantity ofsuch a dry powder needed to deliver an effective dose of calcium ioncannot reasonably be administered to a subject by inhalation.Accordingly, such powders contain calcium ion in an amount that does notproduce therapeutic efficacy.

Suitable magnesium salts that can be present in this type of respirabledry particles of the invention include, for example, magnesium fluoride,magnesium chloride, magnesium bromide, magnesium iodide, magnesiumphosphate, magnesium sulfate, magnesium sulfite, magnesium carbonate,magnesium oxide, magnesium nitrate, magnesium borate, magnesium acetate,magnesium citrate, magnesium gluconate, magnesium maleate, magnesiumsuccinate, magnesium malate, magnesium taurate, magnesium orotate,magnesium glycinate, magnesium naphthenate, magnesium acetylacetonate,magnesium formate, magnesium hydroxide, magnesium stearate, magnesiumhexafluorsilicate, magnesium salicylate or any combination thereof. In apreferred aspect, the dry powder or dry particles include magnesiumsulfate, magnesium lactate, magnesium chloride, magnesium citrate, andmagnesium carbonate. Preferred magnesium salts are magnesium sulfate andmagnesium lactate.

Preferred divalent metal salts (e.g., calcium salts) have one orpreferably two or more of the following characteristics: (i) can beprocessed into a respirable dry particle, (ii) possess sufficientphysicochemical stability in dry powder form to facilitate theproduction of a powder that is dispersible and physically stable over arange of conditions, including upon exposure to elevated humidity, (iii)undergo rapid dissolution upon deposition in the lungs, for example,half of the mass of the cation of the divalent metal can dissolved inless than 30 minutes, less than 15 minutes, less than 5 minutes, lessthan 2 minutes, less than 1 minute, or less than 30 seconds, and (iv) donot possess properties that can result in poor tolerability or adverseevents, such as a significant exothermic or endothermic heat of solution(ΔH) for example, a AH lower than of about −10 kcal/mol or greater thanabout 10 kcal/mol. Rather, a preferred ΔH is between about −9 kcal/moland about 9 kcal/mol, between about −8 kcal/mol and about 8 kcal/mol,between about −7 kcal/mol and about 7 kcal/mol, between about −6kcal/mol and about 6 kcal/mol, between about −5 kcal/mol and about 5kcal/mol, between about −4 kcal/mol and about 4 kcal/mol, between about−3 kcal/mol and about 3 kcal/mol, between about −2 kcal/mol and about 2kcal/mol, between about −1 kcal/mol and about 1 kcal/mol, or about 0kcal/mol.

Suitable divalent metal cation salts (e.g., calcium salts) can havedesired solubility characteristics. In general, highly or moderatelysoluble divalent metal cation salts (e.g., calcium salts) are preferred.For example, suitable divalent metal cation salts (e.g., calcium salts)that are contained in the respirable dry particles and dry powders canhave a solubility in distilled water at room temperature (20-30° C.) and1 bar of at least about 0.4 g/L, at least about 0.85 g/L, at least about0.90 g/L, at least about 0.95 g/L, at least about 1.0 g/L, at leastabout 2.0 g/L, at least about 5.0 g/L, at least about 6.0 g/L, at leastabout 10.0 g/L, at least about 20 g/L, at least about 50 g/L, at leastabout 90 g/L, at least about 120 g/L, at least about 500 g/L, at leastabout 700 g/L or at least about 1000 g/L. Preferably, the divalent metalcation salt has a solubility greater than about 0.90 g/L, greater thanabout 2.0 g/L, or greater than about 90 g/L. Suitable divalent metalcation salts include calcium salts and magnesium salts.

Dry particles and dry powders of the invention can be prepared, ifdesired, that contain divalent metal cation salts (e.g., calcium salts)that are not highly soluble in water. As described herein, such dryparticles and dry powders can be prepared using a feed stock of adifferent, more soluble salt, and permitting anion exchange to producethe desired divalent metal cation salts (e.g., calcium salt) prior to orconcurrently with spray drying. Alternatively, a suspension may also befed to the spray dryer to make respirable dry powders and respirable dryparticles.

Dry powder and particles of the invention may contain a high percentageof active ingredient (e.g., divalent metal cation (e.g., calcium)) inthe composition, and be divalent metal cation dense. The dry particlesmay contain 3% or more, 5% or more, 10% or more, 15% or more, 20% oremore, 25% or more, 30% or more, 35% or more, 40% or more, 50% or more,60% or more, 70% or more, 75% or more, 80% or more, 85% or more. 90% ormore, or 95% or more active ingredient.

It is advantageous when the divalent metal cation salt (e.g., calciumsalt) dissociates to provide two or more moles of divalent metal cation(e.g., Ca²⁺) per mole of salt. Such salts can be used to producerespirable dry powders and dry particles that are dense in divalentmetal cation (e.g., calcium). For example, one mole of calcium citrateprovides three moles of Ca²⁺ upon dissolution. It is also generallypreferred that the divalent metal cation salt (e.g., calcium salt) is asalt with a low molecular weight and/or contain low molecular weightanions. Low molecular weight divalent metal cation salts, such ascalcium salts that contain calcium ions and low molecular weight anions,are divalent cation dense (e.g., Ca²⁺) dense relative to high molecularsalts and salts that contain high molecular weight anions. It isgenerally preferred that the divalent metal cation salt (e.g., calciumsalt) has a molecular weight of less than about 1000 g/mol, less thanabout 950 g/mol, less than about 900 g/mol, less than about 850 g/mol,less than about 800 g/mol, less than about 750 g/mol, less than about700 g/mol, less than about 650 g/mol, less than about 600 g/mol, lessthan about 550 g/mol, less than about 510 g/mol, less than about 500g/mol, less than about 450 g/mol, less than about 400 g/mol, less thanabout 350 g/mol, less than about 300 g/mol, less than about 250 g/mol,less than about 200 g/mol, less than about 150 g/mol, less than about125 g/mol, or less than about 100 g/mol. In addition or alternatively,it is generally preferred that the divalent metal cation (e.g., calciumion) contributes a substantial portion of the weight to the overallweight of the divalent metal cation salt. It is generally preferred thatthe divalent metal cation (e.g., calcium ion) contribute at least 10% ofthe weight of the overall salt, at least 16%, at least 20%, at least24.5%, at least 26%, at least 31%, at least 35%, or at least 38% of theweight of the overall divalent metal cation salt (e.g., calcium salt).

Alternatively or in addition, the respirable dry particles of theinvention can include a suitable divalent metal cation salt (e.g.,calcium salt) that provides divalent metal cation (Ca²⁺), wherein theweight ratio of divalent metal cation (e.g., calcium ion) to the overallweight of said salt is between about 0.1 to about 0.5. For example, theweight ratio of divalent metal cation (e.g, calcium ion) to the overallweight of said salt is between about 0.15 to about 0.5, between about0.18 to about 0.5, between about 0.2 to about 5, between about 0.25 toabout 0.5, between about 0.27 to about 0.5, between about 0.3 to about5, between about 0.35 to about 0.5, between about 0.37 to about 0.5, orbetween about 0.4 to about 0.5.

Alternatively or in addition, the respirable dry particles of theinvention can contain a divalent metal cation salt (e.g., calcium salt)which provides divalent cation (e.g., Ca²⁺) in an amount of at leastabout 5% by weight of the respirable dry particles. For example, therespirable dry particles of the invention can include a divalent metalcation salt (e.g., calcium salt) which provides divalent cation (e.g.,Ca²⁺) in an amount of at least about 7% by weight, at least about 10% byweight, at least about 11% by weight, at least about 12% by weight, atleast about 13% by weight, at least about 14% by weight, at least about15% by weight, at least about 17% by weight, at least about 20% byweight, at least about 25% by weight, at least about 30% by weight, atleast about 35% by weight, at least about 40% by weight, at least about45% by weight, at least about 50% by weight, at least about 55% byweight, at least about 60% by weight, at least about 65% by weight or atleast about 70% by weight of the respirable dry particles.

Alternatively or in addition, the respirable dry particles of theinvention can contain a divalent metal cation salt which providesdivalent metal cation (e.g., Ca²⁺, Be²⁺, Mg²⁺, Sr²⁺, Ba²⁺, Fe²⁺) in anamount of at least about 5% by weight of the respirable dry particlesand also contain a monovalent salt (e.g, sodium salt, lithium salt,potassium salt) which provides monovalent cation (e.g, Na⁺, Li⁺, K⁺) inan amount of at least about 3% by weight of the respirable dryparticles. For example, the respirable dry particles of the inventioncan include a divalent metal cation salt (e.g., calcium salt) whichprovides divalent cation (e.g., Ca²⁺) in an amount of at least about 7%by weight, at least about 10% by weight, at least about 11% by weight,at least about 12% by weight, at least about 13% by weight, at leastabout 14% by weight, at least about 15% by weight, at least about 17% byweight, at least about 20% by weight, at least about 25% by weight, atleast about 30% by weight, at least about 35% by weight, at least about40% by weight, at least about 45% by weight, at least about 50% byweight, at least about 55% by weight, at least about 60% by weight, atleast about 65% by weight or at least about 70% by weight of therespirable dry particles; and further contain a monovalent salt sodiumsalt which provides monovalent anion (Na⁺) in an amount of at leastabout 3%, at least about 4%, at least about 5%, at least about 6%, atleast about 7%, at least about 8%, at least about 9%, at least about10%, at least about 11%, at least about 12%, at least about 14%, atleast about 16%, at least about 18%, at least about 20%, at least about22%, at least about 25%, at least about 27%, at least about 29%, atleast about 32%, at least about 35%, at least about 40%, at least about45%, at least about 50% or at least about 55% by weight of therespirable dry particles.

Alternatively or in addition, the respirable dry particles of theinvention contain a divalent metal cation salt and a monovalent cationsalt, where the divalent cation, as a component of one or more salts, ispresent in an amount of at least 5% by weight of dry particle, and theweight ratio of divalent cation to monovalent cation is about 50:1(i.e., about 50 to about 1) to about 0.1:1 (i.e., about 0.1 to about 1).The weight ratio of divalent metal cation to monovalent cation, is basedon the amount of divalent metal cation and monovalent cation that arecontained in the divalent metal cation salt and monovalent salts,respectively, that are contained in the dry particle. In particularexamples, the weight ratio of divalent metal cation to monovalent cationis about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1,about 0.7:1, about 0.8:1, about 0.86:1, about 0.92:1, about 1:1; about1.3:1, about 2:1, about 5:1, about 10:1, about 15:1, about 20:1, about25:1, about 30:1, about 35:1, about 40:1, about 45:1, or about 50:1,about 20:1 to about 0.1:1, about 15:1 to about 0.1:1, about 10:1 toabout 0.1:1, or about 5:1 to about 0.1:1.

Alternatively or in addition, the respirable dry particles of theinvention can contain a divalent metal cation salt and a monovalentcation salt, in which the divalent metal cation salt and the monovalentcation salt contain chloride, lactate, citrate or sulfate as the counterion, and the ratio of divalent metal cation (e.g., Ca²⁺, Be²⁺, Mg²⁺,Sr²⁺, Ba²⁺, Fe²⁺) to monovalent cation (e.g, Na⁺, Li⁺, K⁺) mole:mole isabout 50:1 (i.e., about 50 to about 1) to about 0.1:1 (i.e., about 0.1to about 1). The mole ratio of divalent metal cation to monovalentcation, is based on the amount of divalent metal cation and monovalentcation that are contained in the divalent metal cation salt andmonovalent cation salt, respectively, that are contained in the dryparticle. Preferably, divalent metal cation, as a component of one ormore divalent metal cation salts, is present in an amount of at least 5%by weight of the respirable dry particle. In particular examples,divalent metal cation and monovalent cation are present in therespirable dry particles in a mole ratio of about 8.0:1, about 7.5:1,about 7.0:1, about 6.5:1, about 6.0:1, about 5.5:1, about 5.0:1, about4.5:1, about 4.0:1, about 3.5:1, about 3.0:1, about 2.5:1, about 2.0:1,about 1.5:1, about 1.0:1, about 0.77:1, about 0.65:1, about 0.55:1,about 0.45:1, about 0.35:1, about 0.25:1, or about 0.2:1, about 8.0:1 toabout 0.55:1, about 7.0:1 to about 0.55:1, about 6.0:1 to about 0.55:1,about 5.0:1 to about 0.55:1, about 4.0:1 to about 0.55:1, about 3.0:1 toabout 0.55:1, about 2.0:1 to about 0.55:1, or about 1.0:1 to about0.55:1.

Preferably, the ratio of divalent metal cation (e.g., Ca²⁺, Be²⁺, Mg²⁺,Sr²⁺, Ba²⁺, Fe²⁺) to monovalent cation (e.g, Na⁺, Li⁺, K⁺) mole:mole isabout 16.0:1.0 to about 1.0:1.0, about 16.0:1.0 to about 2.0:1.0, about8.0:1.0 to about 1.0:1.0, about 4.0:1.0 to about 1.0:1.0, about 4:0:1.0to about 2.0:1.0. More preferably, the divalent metal cation andmonovalent cation are present in the respirable dry particles in a moleratio of about 8.0:1.0 to about 2.0:1.0 or about 4.0:1.0 to about2.0:1.0. Most preferably, the divalent metal cation is Ca²⁺ and themonovalent cation is Na⁺.

Preferred respirable dry particles contain at least one calcium saltselected from the group consisting of calcium lactate, calcium citrate,calcium sulfate, and calcium chloride, and also contain sodium chloride.

Calcium citrate, calcium sulfate and calcium lactate possess sufficientaqueous solubility to allow for their processing into respirable drypowders via spray-drying and to facilitate their dissolution upondeposition in the lungs, yet possess a low enough hygroscopicity toallow for the production of dry powders with high calcium salt loadsthat are relatively physically stable upon exposure to normal andelevated humidity. Calcium citrate, calcium sulfate and calcium lactatealso have a significantly lower heat of solution than calcium chloride,which is beneficial for administration to the respiratory tract, andcitrate, sulfate and lactate ions are safe and acceptable for inclusionin pharmaceutical compositions.

Accordingly, in addition to any combination of the features andproperties described herein, the respirable dry particles of theinvention can contain one or more salts in a total amount of at leastabout 51% by weight of the respirable dry particles; wherein each of theone or more salts independently consists of a cation selected from thegroup consisting of calcium and sodium and an anion selected from thegroup consisting of lactate (C₃H₅O₃ ⁻), chloride (CE) citrate (C₆H₅O₇³⁻) and sulfate (SO₄ ²⁻), with the proviso that at least one of thesalts is a calcium salt. For example, the respirable dry particles ofthe invention can include one or more of the salts in a total amount ofat least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, or at leastabout 95% by weight of the respirable dry particles.

Alternatively or in addition, the respirable dry particles of theinvention can contain a calcium salt and a sodium salt, where thecalcium cation, as a component of one or more calcium salts, is presentin an amount of at least 5% by weight of the dry particle, and theweight ratio of calcium ion to sodium ion is about 50:1 (i.e., about 50to about 1) to about 0.1:1 (i.e., about 0.1 to about 1). The weightratio of calcium ion to sodium ion, is based on the amount of calciumion and sodium ion that are contained in the calcium salt and sodiumsalts, respectively, that are contained in the dry particle. Inparticular examples, the weight ratio of calcium ion to sodium ion isabout 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about0.7:1, about 0.8:1, about 0.86:1, about 0.92:1, about 1:1; about 1.3:1,about 2:1, about 5:1, about 10:1, about 15:1, about 20:1, about 25:1,about 30:1, about 35:1, about 40:1, about 45:1, or about 50:1, about20:1 to about 0.1:1, about 15:1 to about 0.1:1, about 10:1 to about0.1:1, or about 5:1 to about 0.1:1.

Alternatively or in addition, the respirable dry particles of theinvention can contain a calcium salt and a sodium salt, in which thecalcium salt and the sodium salt contain chloride, lactate, citrate orsulfate as the counter ion, and the ratio of calcium to sodium mole:moleis about 50:1 (i.e., about 50 to about 1) to about 0.1:1 (i.e., about0.1 to about 1). The mole ratio of calcium to sodium, is based on theamount of calcium and sodium that are contained in the calcium salt andsodium salt, respectively, that are contained in the dry particle.Preferably, calcium, as a component of one or more calcium salts, ispresent in an amount of at least 5% by weight of the respirable dryparticle. In particular examples, calcium and sodium are present in therespirable dry particles in a mole ratio of about 8.0:1, about 7.5:1,about 7.0:1, about 6.5:1, about 6.0:1, about 5.5:1, about 5.0:1, about4.5:1, about 4.0:1, about 3.5:1, about 3.0:1, about 2.5:1, about 2.0:1,about 1.5:1, about 1.0:1, about 0.77:1, about 0.65:1, about 0.55:1,about 0.45:1, about 0.35:1, about 0.25:1, or about 0.2:1, about 8.0:1 toabout 0.55:1, about 7.0:1 to about 0.55:1, about 6.0:1 to about 0.55:1,about 5.0:1 to about 0.55:1, about 4.0:1 to about 0.55:1, about 3.0:1 toabout 0.55:1, about 2.0:1 to about 0.55:1, or about 1.0:1 to about0.55:1.

If desired, the respirable dry particles described herein can include aphysiologically or pharmaceutically acceptable carrier or excipient. Forexample, a pharmaceutically-acceptable excipient includes any of thestandard carbohydrate, sugar alcohol, and amino acid carriers that areknown in the art to be useful excipients for inhalation therapy, eitheralone or in any desired combination. These excipients are generallyrelatively free-flowing particulates, do not thicken or polymerize uponcontact with water, are toxicologically innocuous when inhaled as adispersed powder and do not significantly interact with the active agentin a manner that adversely affects the desired physiological action ofthe salts of the invention. Carbohydrate excipients that are useful inthis regard include the mono- and polysaccharides. Representativemonosaccharides include carbohydrate excipients such as dextrose(anhydrous and the monohydrate; also referred to as glucose and glucosemonohydrate), galactose, mannitol, D-mannose, sorbose and the like.Representative disaccharides include lactose, maltose, sucrose,trehalose and the like. Representative trisaccharides include raffinoseand the like. Other carbohydrate excipients include maltodextrin andcyclodextrins, such as 2-hydroxypropyl-beta-cyclodextrin can be used asdesired. Representative sugar alcohols include mannitol, sorbitol andthe like.

Suitable amino acid excipients include any of the naturally occurringamino acids that form a powder under standard pharmaceutical processingtechniques and include the non-polar (hydrophobic) amino acids and polar(uncharged, positively charged and negatively charged) amino acids, suchamino acids are of pharmaceutical grade and are generally regarded assafe (GRAS) by the U.S. Food and Drug Administration. Representativeexamples of non-polar amino acids include alanine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan and valine.Representative examples of polar, uncharged amino acids include cystine,glycine, glutamine, serine, threonine, and tyrosine. Representativeexamples of polar, positively charged amino acids include arginine,histidine and lysine. Representative examples of negatively chargedamino acids include aspartic acid and glutamic acid. These amino acidsare generally available from commercial sources that providepharmaceutical-grade products such as the Aldrich Chemical Company,Inc., Milwaukee, Wis. or Sigma Chemical Company, St. Louis, Mo.

Preferred amino acid excipients, such as the hydrophobic amino acidleucine, can be present in the dry particles of the invention in anamount of about 74% or less by weight of respirable dry particles. Forexample, the respirable dry particles of the invention can contain theamino acid leucine in an amount of about 5% to about 30% by weight,about 10% to about 20% by weight, about 5% to about 20% by weight, about50% or less by weight, about 45% or less by weight, about 40% or less byweight, about 35% or less by weight, about 30% or less by weight, about25% or less by weight, about 20% or less by weight, about 18% or less byweight, about 16% or less by weight, about 15% or less by weight, about14% or less by weight, about 13% or less by weight, about 12% or less byweight, about 11% or less by weight, about 10% or less by weight, about9% or less by weight, about 8% or less by weight, about 7% or less byweight, about 6% or less by weight, about 5% or less by weight, about 4%or less by weight, about 3% or less by weight, about 2% or less byweight, or about 1% or less by weight.

Preferred carbohydrate excipients, such as maltodextrin and mannitol,can be present in the dry particles of the invention in an amount ofabout 74% or less by weight of respirable dry particles. For example,the respirable dry particles of the invention can contain maltodextrinin an amount of about 50% or less by weight, about 45% or less byweight, about 40% or less by weight, about 35% or less by weight, about30% or less by weight, about 25% or less by weight, about 20% or less byweight, about 18% or less by weight, about 16% or less by weight, about15% or less by weight, about 14% or less by weight, about 13% or less byweight, about 12% or less by weight, about 11% or less by weight, about10% or less by weight, about 9% or less by weight, about 8% or less byweight, about 7% or less by weight, about 6% or less by weight, about 5%or less by weight, about 4% or less by weight, about 3% or less byweight, about 2% or less by weight, or about 1% or less by weight. Insome preferred aspects, the dry particles contain an excipient selectedfrom leucine, maltodextrin, mannitol and any combination thereof. Inparticular embodiments, the excipient is leucine, maltodextrin, ormannitol.

In particular embodiments, the respirable dry particles of the inventioncan contain (a) a calcium salt selected from calcium lactate, calciumcitrate or calcium sulfate in an amount of at least about 30%, at leastabout 40%, at least about 45% by weight, or at least about 50% by weightof dry particle; and (b) a sodium salt, such as sodium chloride, in anamount of at least about 25% or at least about 30% by weight of dryparticle, and have any of the properties or features described herein.If desired, an excipient, such as leucine, maltodextrin, mannitol or anycombination thereof, can be present an amount of about 74% or less orabout 50% or less or about 20% or less by weight of the dry particle.For example, the respirable dry particles of the invention can include(a) a calcium salt in an amount of about 30% to about 65%, about 40% toabout 65%, or about 45% to about 65% by weight of dry particle; (b) asodium salt, such as sodium chloride, in an amount of about 25% to about60%, or about 30% to about 60% by weight of dry particle; (c) anexcipient, such as leucine, maltodextrin, mannitol or any combinationthereof, in an amount of about 20% or less by weight of dry particle, ormore preferably about 10% or less by weight of dry particle, and (d)have any of the properties or features, such as 1/4 bar, 0.5/4 bar,VMGD, MMAD, FPF described herein.

In other embodiments, the respirable dry particles of the invention cancontain (a) a calcium salt selected from calcium lactate, calciumcitrate or calcium sulfate in an amount of at least about 30%, at leastabout 40%, at least about 45% by weight, or at least about 50% by weightof dry particle; and (b) a sodium salt, such as sodium chloride, in anamount between about 2% and about 20%, or between about 3.5% and about10% by weight of dry particle, and have any of the properties orfeatures described herein. If desired, an excipient, such as leucine,maltodextrin, mannitol or any combination thereof, or the like, can bepresent in an amount of about 74% or less or about 50% or less or about20% or less by weight of the dry particle. For example, the respirabledry particles of the invention can include (a) a calcium salt in anamount of about 30% to about 65%, about 40% to about 65%, or about 45%to about 65% by weight of dry particle; (b) a sodium salt, such assodium chloride, in an amount between about 2% and about 20%, or betweenabout 3.5% and about 10% by weight of dry particle; (c) an excipient,such as leucine, maltodextrin, mannitol or any combination thereof, inan amount of about 20% or less by weight of dry particle, or morepreferably about 10% or less by weight of dry particle, and (d) have anyof the properties or features, such as 1/4 bar, 0.5/4 bar, VMGD, MMAD,FPF described herein.

In some aspects, the respirable dry particles comprise a divalent metalion salt and a monovalent salt and are characterized by the crystallineand amorphous content of the particles. For example, the respirable dryparticles can comprise a mixture of amorphous and crystalline content,such as an amorphous divalent metal ion salt-rich phase and acrystalline monovalent salt or excipient phase. Respirable dry particlesof this type provide several advantages. For example as describedherein, the crystalline phase (e.g. crystalline sodium chloride and/orcrystalline leucine) can contribute to the stability of the dry particlein the dry state and to the dispersibility characteristics, whereas theamorphous phase (e.g., amorphous calcium salt) can facilitate rapidwater uptake and dissolution of the particle upon deposition in therespiratory tract. It is particularly advantageous when salts withrelatively high aqueous solubilities (such as sodium chloride) that arepresent in the dry particles are in a crystalline state and when saltswith relatively low aqueous solubilities (such as calcium citrate) arepresent in the dry particles in an amorphous state.

The amorphous phase is also characterized by a high glass transitiontemperature (T_(g)), such as a T_(g) of at least 90° C., at least 100°C., at least 110° C., at least 120° C., at least 125° C., at least 130°C., at least 135° C., at least 140° C., between 120° C. and 200° C.,between 125° C. and 200° C., between 130° C. and 200° C., between 120°C. and 190° C., between 125° C. and 190° C., between 130° C. and 190°C., between 120° C. and 180° C., between 125° C. and 180° C., or between130° C. and 180° C.

In some embodiments, the respirable dry particles contain divalent metalcation salt-rich amorphous phase and a monovalent salt crystalline phaseand the ratio of amorphous phase to crystalline phase (w:w) is about5:95 to about 95:5, about 5:95 to about 10:90, about 10:90 to about20:80, about 20:80 to about 30:70, about 30:70 to about 40:60, about40:60 to about 50:50; about 50:50 to about 60:40, about 60:40 to about70:30, about 70:30 to about 80:20, or about 90:10 to about 95:5. Inother embodiments, the respirable dry particles contain divalent metalcation salt-rich amorphous phase and a monovalent salt crystalline phaseand the ratio of amorphous phase to particle by weight (w:w) is about5:95 to about 95:5, about 5:95 to about 10:90, about 10:90 to about20:80, about 20:80 to about 30:70, about 30:70 to about 40:60, about40:60 to about 50:50; about 50:50 to about 60:40, about 60:40 to about70:30, about 70:30 to about 80:20, or about 90:10 to about 95:5. Inother embodiments, the respirable dry particles contain divalent metalcation salt-rich amorphous phase and a monovalent salt crystalline phaseand the ratio of crystalline phase to particle by weight (w:w) is about5:95 to about 95:5, about 5:95 to about 10:90, about 10:90 to about20:80, about 20:80 to about 30:70, about 30:70 to about 40:60, about40:60 to about 50:50; about 50:50 to about 60:40, about 60:40 to about70:30, about 70:30 to about 80:20, or about 90:10 to about 95:5.

In some embodiments, the respirable dry particles comprise a calciumsalt, such as calcium citrate, calcium sulfate, calcium lactate, calciumchloride or any combination thereof, and a sodium salt, such as sodiumchloride, sodium citrate, sodium sulfate, sodium lactate, or anycombination thereof, wherein the respirable dry particle contains acalcium salt-rich amorphous phase, and a crystalline sodium salt phase.In particular embodiments, the calcium salt-rich amorphous phaseincludes calcium citrate and at least some calcium chloride, calciumlactate and at least some calcium chloride, or calcium sulfate and atleast some calcium chloride. In some embodiments, the respirable dryparticles contain calcium salt-rich amorphous phase and a sodium saltcrystalline phase and the ratio of amorphous phase to crystalline phase(w:w) is about 5:95 to about 95:5, about 5:95 to about 10:90, about10:90 to about 20:80, about 20:80 to about 30:70, about 30:70 to about40:60, about 40:60 to about 50:50; about 50:50 to about 60:40, about60:40 to about 70:30, about 70:30 to about 80:20, or about 90:10 toabout 95:5. In other embodiments, the respirable dry particles containcalcium salt-rich amorphous phase and a sodium salt crystalline phaseand the ratio of amorphous phase to particle by weight (w:w) is about5:95 to about 95:5, about 5:95 to about 10:90, about 10:90 to about20:80, about 20:80 to about 30:70, about 30:70 to about 40:60, about40:60 to about 50:50; about 50:50 to about 60:40, about 60:40 to about70:30, about 70:30 to about 80:20, or about 90:10 to about 95:5. Inother embodiments, the respirable dry particles contain calciumsalt-rich amorphous phase and a sodium salt crystalline phase and theratio of crystalline phase to particle by weight (w:w) is about 5:95 toabout 95:5, about 5:95 to about 10:90, about 10:90 to about 20:80, about20:80 to about 30:70, about 30:70 to about 40:60, about 40:60 to about50:50; about 50:50 to about 60:40, about 60:40 to about 70:30, about70:30 to about 80:20, or about 90:10 to about 95:5.

Preferrably, the respirable dry particles have a 1/4 bar or 0.5/4 bar of2 or less, as described herein. For example, a 1/4 bar or 0.5/4 bar of1.9 or less, 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 orless, 1.3 or less, 1.2 or less, 1.1 or less or about 1.0. Alternativelyor in addition, the respirable dry particles have an MMAD of about 5microns or less. Alternatively or in addition, the respirable dryparticles can have a VMGD between about 0.5 microns and about 5 microns,or a VMGD between about 5 microns and about 20 microns. Alternatively orin addition, the respirable dry particles can have a heat of solutionthat not is greater than about −10 kcal/mol (e.g., between −10 kcal/moland 10 kcal/mol).

As described herein, the respirable dry particles can further comprisean excipient, such as leucine, maltodextrin or mannitol. The excipientcan be crystalline or amorphous or present in a combination of theseforms. In some embodiments, the excipient is amorphous or predominatelyamorphous. In some embodiments, the respirable dry particles aresubstantially crystalline.

As described herein, surface RAMAN mapping spectra of respirable drypowders that contained an excipient (i.e., leucine, maltodextrin)indicate that the excipients were not concentrated at the surface of theparticles, and that the excipients are either evenly distributedthroughout the particle or not exposed to the surface of the particle.Leucine excipients, in particular, have been reported to improvedispersibility when concentrated on the surface of particles. See, e.g.,US2003/0186894. Accordingly, it does not appear that leucine is actingas a dispersion enhancer in this way. Thus, in the respirable dryparticles of the invention that contain an excipient (e.g., leucine),the excipient can be distributed within the particle but not on theparticle surface, or distributed throughout the particle (e.g.,homogenously distributed). For example, in some particular embodiments,a respirable dry particle of the invention does not produce acharacteristic peak indicative of the presence of an excipient (e.g.,leucine) under RAMAN spectroscopy. In more particular embodiments, a dryrespirable powder that contains leucine does not produce acharacteristic leucine peak (e.g., at 1340 cm⁻¹) under RAMANspectroscopy.

As described herein, some powders of the invention have poor flowproperties. Yet, surprisingly, these powders are highly dispersible.This is surprising because flow properties and dispersibility are bothknown to be negatively affected by particle agglomeration oraggregation. Thus, it was unexpected that particles that have poor flowcharacteristics would be highly dispersible.

In addition to any of the features and properties described herein, inany combination, the respirable dry particles can have poor flowproperties yet have good dispersibility. For example, the respirable dryparticles can have a Hausner Ratio that is greater than 1.35 (e.g, 1.4or greater, 1.5 or greater, 1.6 or greater, 1.7 or greater, 1.8 orgreater, 1.9 or greater, 2.0 or greater) and also have a 1/4 bar or0.5/4 bar that is 2 or less, 1.9 or less, 1.8 or less, 1.7 or less, 1.6or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or lessor about 1.0.

In addition to any of the features and properties described herein, inany combination, the respirable dry particles can have a heat ofsolution that is not highly exothermic. Preferably, the heat of solutionis determined using the ionic liquid of a simulated lung fluid (e.g. asdescribed in Moss, O. R. 1979. Simulants of lung interstitial fluid.Health Phys. 36, 447-448; or in Sun, G. 2001. Oxidative interactions ofsynthetic lung epithelial lining fluid with metal-containing particulatematter. Am J Physiol Lung Cell Mol Physiol. 281, L807-L815) at pH 7.4and 37° C. in an isothermal calorimeter. For example, the respirable dryparticles can have a heat of solution that is less exothermic than theheat of solution of calcium chloride dihydrate, e.g., have a heat ofsolution that is greater than about −10 kcal/mol, greater than about −9kcal/mol, greater than about −8 kcal/mol, greater than about −7kcal/mol, greater than about −6 kcal/mol, greater than about −5kcal/mol, greater than about −4 kcal/mol, greater than about −3kcal/mol, greater than about −2 kcal/mol, greater than about −1 kcal/molor about −10 kcal/mol to about 10 kcal/mol. The respirable dry particlescan have a heat of solution of about −8 kcal/mol to about 8 kcal/mol,about −6 kcal/mol to about 6 kcal/mol, or about −4 kcal/mol to about 4kcal/mol.

If desired, the salt formulation can include one or more additionalagents, such as mucoactive or mucolytic agents, surfactants,antibiotics, antivirals, antihistamines, cough suppressants,bronchodilators, anti-inflammatory agents, steroids, vaccines,adjuvants, expectorants, macromolecules, or therapeutics that arehelpful for chronic maintenance of cystic fibrosis (CF). The additionalagent can be blended with a dry powder of the salt formulation orco-spray dried as desired.

In some embodiments, the salt formulation can contain an agent thatdisrupts and/or disperse biofilms. Suitable examples of agents topromote disruption and/or dispersion of biofilms include specific aminoacid stereoisomers, e.g. D-leucine, D-methionine, D-tyrosine,D-tryptophan, and the like. (Kolodkin-Gal, I., D. Romero, et al.“D-amino acids trigger biofilm disassembly.” Science 328(5978):627-629.) For example, all or a portion of the leucine in the drypowders described herein which contain leucine can be D-leucine.

Examples of suitable mucoactive or mucolytic agents include MUC5AC andMUC5B mucins, DNA-ase, N-acetylcysteine (NAC), cysteine, nacystelyn,dornase alfa, gelsolin, heparin, heparin sulfate, P2Y2 agonists (e.g.UTP, INS365), nedocromil sodium, hypertonic saline, and mannitol.

Suitable surfactants include L-alpha-phosphatidylcholine dipalmitoyl(“DPPC”), diphosphatidyl glycerol (DPPG),1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS),1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC),1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1-palmitoyl-2-oleoylphosphatidylcholine (POPC), fatty alcohols,polyoxyethylene-9-lauryl ether, surface active fatty, acids, sorbitantrioleate (Span 85), glycocholate, surfactin, poloxomers, sorbitan fattyacid esters, tyloxapol, phospholipids, and alkylated sugars.

If desired, the salt formulation can contain an antibiotic. Theantibiotic can be suitable for treating any desired bacterial infection,and salt formulations that contain an antibiotic can be used to reducethe spread of infection, either within a patient or from patient topatient. For example, salt formulations for treating bacterial pneumoniaor VAT, can further comprise an antibiotic, such as a macrolide (e.g.,azithromycin, clarithromycin and erythromycin), a tetracycline (e.g.,doxycycline, tigecycline), a fluoroquinolone (e.g., gemifloxacin,levofloxacin, ciprofloxacin and mocifloxacin), a cephalosporin (e.g.,ceftriaxone, defotaxime, ceftazidime, cefepime), a penicillin (e.g.,amoxicillin, amoxicillin with clavulanate, ampicillin, piperacillin, andticarcillin) optionally with a β-lactamase inhibitor (e.g., sulbactam,tazobactam and clavulanic acid), such as ampicillin-sulbactam,piperacillin-tazobactam and ticarcillin with clavulanate, anaminoglycoside (e.g., amikacin, arbekacin, gentamicin, kanamycin,neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin,tobramycin, and apramycin), a penem or carbapenem (e.g. doripenem,ertapenem, imipenem and meropenem), a monobactam (e.g., aztreonam), anoxazolidinone (e.g., linezolid), vancomycin, glycopeptide antibiotics(e.g. telavancin), tuberculosis-mycobacterium antibiotics and the like.

If desired, the salt formulation can contain an agent for treatinginfections with mycobacteria, such as Mycobacterium tuberculosis.Suitable agents for treating infections with mycobacteria (e.g., M.tuberculosis) include an aminoglycoside (e.g. capreomycin, kanamycin,streptomycin), a fluoroquinolone (e.g. ciprofloxacin, levofloxacin,moxifloxacin), isozianid and isozianid analogs (e.g. ethionamide),aminosalicylate, cycloserine, diarylquinoline, ethambutol, pyrazinamide,protionamide, rifampin, and the like.

If desired, the salt formulation can contain a suitable antiviral agent,such as oseltamivir, zanamavir, amantidine, rimantadine, ribavirin,gancyclovir, valgancyclovir, Foscavir, Cytogam® (Cytomegalovirus ImmuneGlobulin), pleconaril, rupintrivir, palivizumab, motavizumab,cytarabine, docosanol, denotivir, cidofovir, and acyclovir. The saltformulation can contain a suitable anti-influenza agent, such aszanamivir, oseltamivir, amantadine, or rimantadine.

Suitable antihistamines include clemastine, asalastine, loratadine,fexofenadine and the like.

Suitable cough suppressants include benzonatate, benproperine,clobutinal, diphenhydramine, dextromethorphan, dibunate, fedrilate,glaucine, oxalamine, piperidione, opiods such as codeine and the like.

Suitable brochodilators include short-acting beta₂ agonists, long-actingbeta₂ agonists (LABA), long-acting muscarinic anagonists (LAMA),combinations of LABAs and LAMAs, methylxanthines, short-actinganticholinergic agents (may also be referred to as short actinganti-muscarinic), long-acting bronchodilators and the like.

Suitable short-acting beta₂ agonists include albuterol, epinephrine,pirbuterol, levalbuterol, metaproteronol, maxair, and the like.

Examples of albuterol sulfate formulations (also called salbutamol)include Inspiryl (AstraZeneca Plc), Salbutamol SANDOZ (Sanofi-Aventis),Asmasal clickhalcr (Vectura Group Plc.), Ventolin® (GlaxoSmithKlinePlc), Salbutamol GLAND (GlaxoSmithKline Plc), Airomir® (TevaPharmaceutical Industries Ltd.), ProAir IIFA (Teva PharmaceuticalIndustries Ltd.), Salamol (Teva Pharmaceutical Industries Ltd.), Ipramol(Teva Pharmaceutical Industries Ltd), Albuterol sulfate TEVA (TevaPharmaceutical Industries Ltd), and the like. Examples of epinephrineinclude Epinephine Mist KING (King Pharmaceuticals, Inc.), and the like.Examples of pirbuterol as pirbuterol acetate include Maxair® (TevaPharmaceutical Industries Ltd.), and the like. Examples of levalbuterolinclude Xopenex® (Sepracor), and the like. Examples of metaproteronolformulations as metaproteronol sulfate include Alupent® (BoehringerIngelheim GmbH), and the like.

Suitable LABAs include salmeterol, formoterol and isomers (e.g.arformoterol), clenbuterol, tulobuterol, vilanterol (Revolair™),indacaterol, carmoterol, isoproterenol, procaterol, bambuterol,milveterol, olodaterol and the like.

Examples of salmeterol formulations include salmeterol xinafoate asSerevent® (GlaxoSmithKline Plc), salmeterol as Inaspir (LaboratoriosAlmirall, S.A.), Advair® HFA (GlaxoSmithKline PLC), Advair Diskus®(GlaxoSmithKline PLC, Theravance Inc), Plusvent (Laboratorios Almirall,S.A.), VR315 (Novartis, Vectura Group PLC) and the like. Examples offormoterol and isomers (e.g., arformoterol) include Foster (ChiesiFarmaceutici S.p.A), Atimos (Chiesi Farmaceutici S.p.A, NycomedIntemaional Management), Flutiform® (Abbott Laboratories, SkyePharmaPLC), MFF258 (Novartis AG), Formoterol clickhaler (Vectura Group PLC),Formoterol HFA (SkyePharma PLC), Oxis® (Astrazeneca PLC), Oxis pMDI(Astrazeneca), Foradil® Aerolizer (Novartis, Schering-Plough Corp,Merck), Foradil® Certihaler (Novartis, SkyePharma PLC), Symbicort®(AstraZeneca), VR632 (Novartis AG, Sandoz International GmbH), MFF258(Merck & Co Inc, Novartis AG), Alvesco® Combo (Nycomed InternationalManagement GmbH, Sanofi-Aventis, Sepracor Inc), Mometasone furoate(Schering-Plough Corp), and the like. Examples of clenbuterol includeVentipulmin® (Boehringer Ingelheim), and the like. Examples oftulobuterol include Hokunalin Tape (Abbott Japan Co., Ltd., Maruho Co.,Ltd.), and the like. Examples of vilanterol include Revolair™(GlaxoSmithKline PLC), GSK64244 (GlaxoSmithKline PLC), and the like.Examples of indacaterol include QAB149 (Novartis AG, SkyePharma PLC),QMF149 (Merck & Co Inc) and the like. Examples of carmoterol includeCHF4226 (Chiese Farmaceutici S.p.A., Mitsubishi Tanabe PharmaCorporation), CHF5188 (Chiesi Farmaceutici S.p.A), and the like.Examples of isoproterenol sulfate include Aludrin (Boehringer IngelheimGmbH) and the like. Examples of procaterol include Meptin clickhaler(Vectura Group PLC), and the like. Examples of bambuterol include Bambec(AstraZeneca PLC), and the like. Examples of milveterol includeGSK159797C (GlaxoSmithKline PLC), TD3327 (Theravance Inc), and the like.Examples of olodaterol include BI1744CL (Boehringer Ingelheim GmbH) andthe like.

Examples of LAMAs include tiotroprium (Spiriva), trospium chloride,glycopyrrolate, aclidinium, ipratropium and the like.

Examples of tiotroprium formulations include Spiriva®(Boehringcr-Ingleheim, Pfizer), and the like. Examples of glycopyrrolateinclude Robinul® (Wyeth-Ayerst), Robinul® Forte (Wyeth-Ayerst), NVA237(Novartis), and the like. Examples of aclidinium include Eklira® (ForestLabaoratories, Almirall), and the like.

Examples of combinations of LABAs and LAMAs include indacaterol withglycopyrrolate, formoterol with glycopyrrolate, indacaterol withtiotropium, olodaterol and tiotropium, vilanterol with a LAMA, and thelike.

Examples of combinations of indacaterol with glycopyrrolate includeQVA149A (Novartis), and the like. Examples of combinations of formoterolwith glycopyrrolate include PT003 (Pearl Therapeutics) and the like.Examples of combinations of olodaterol with tiotropium include BI1744with Spirva (Boehringer Ingelheim) and the like. Examples ofcombinations of vilanterol with a LAMA include GSK573719 with GSK642444(GlaxoSmithKline PLC), and the like.

Examples of methylxanthine include aminophylline, ephedrine,theophylline, oxtriphylline, and the like.

Examples of aminophylline formulations include Aminophylline BOEHRINGER(Boehringer Ingelheim GmbH) and the like. Examples of ephedrine includeBronkaid® (Bayer AG), Broncholate (Sanofi-Aventis), Primatene® (Wyeth),Tedral SA®, Marax (Pfizer Inc) and the like. Examples of theophyllineinclude Euphyllin (Nycomed International Management GmbH), Theo-dur(Pfizer Inc, Teva Pharmaceutical Industries Ltd) and the like. Examplesof oxtriphylline include Choledyl SA (Pfizer Inc) and the like.

Examples of short-acting anticholinergic agents include ipratropiumbromide, and oxitropium bromide.

Examples of ipratropium bromide formulations includeAtrovent®/Apovent/Inpratropio (Boehringer Ingelheim GmbH), Ipramol (TevaPharmaceutical Industries Ltd) and the like. Examples of oxitropiumbromide include Oxivent (Boehringer Ingelheim GmbH), and the like.

Suitable anti-inflammatory agents include leukotriene inhibitors,phosphodiesterase 4 (PDE4) inhibitors, other anti-inflammatory agents,and the like.

Suitable leukotriene inhibitors include montelukast (cystinylleukotriene inhibitors), masilukast, zafirleukast (leukotriene D4 and E4receptor inhibitors), pranlukast, zileuton (5-lipoxygenase inhibitors),and the like.

Examples of montelukast formulations (cystinyl leukotriene inhibitor)include Singulair® (Merck & Co Inc), Loratadine, montelukast sodiumSCHERING (Schering-Plough Corp), MK0476C (Merck & Co Inc), and the like.Examples of masilukast include MCC847 (AstraZeneca PLC), and the like.Examples of zafirlukast (leukotriene D4 and E4 receptor inhibitor)include Accolate® (AstraZeneca PLC), and the like. Examples ofpranlukast include Azlaire (Schering-Plough Corp). Examples of zileuton(5-LO) include Zyflo® (Abbott Laboratories), Zyflo CR® (AbbottLaboratories, SkyePharma PLC), Zileuton ABBOTT LABS (AbbottLaboratories), and the like. Suitable PDE4 inhibitors includecilomilast, roflumilast, oglemilast, tofimilast, and the like.

Examples of cilomilast formulations include Ariflo (GlaxoSmithKlinePLC), and the like. Examples of roflumilast include Daxas® (NycomedInternational Management GmbH, Pfizer Inc), APTA2217 (Mitsubishi TanabePharma Corporation), and the like. Examples of oglemilast includeGRC3886 (Forest Laboratories Inc), and the like. Examples of tofimilastinclude Tofimilast PFIZER INC (Pfizer Inc), and the like.

Other anti-inflammatory agents include omalizumab (anti-IgEimmunoglobulin Daiichi Sankyo Company, Limited), Zolair (anti-IgEimmunoglobulin, Genentech Inc, Novartis AG, Roche Holding Ltd), Solfa(LTD4 antagonist and phosphodiesterase inhibitor, Takeda PharmaceuticalCompany Limited), IL-13 and IL-13 receptor inhibitors (such as AMG-317,MILR1444A, CAT-354, QAX576, IMA-638, Anrukinzumab, IMA-026, MK-6105,DOM-0910, and the like), IL-4 and IL-4 receptor inhibitors (such asPitrakinra, AER-003, AIR-645, APG-201, DOM-0919, and the like), IL-1inhibitors such as canakinumab, CRTh2 receptor antagonists such asAZD1981 (CRTh2 receptor antagonist, AstraZeneca), neutrophil elastaseinhibitor such as AZD9668 (neutrophil elastase inhibitor, fromAstraZeneca), GW856553X Losmapimod (P38 kinase inhibitor,GlaxoSmithKline PLC), Arofylline LAB ALMIRALL (PDE-4 inhibitor,Laboratorios Almirall, S.A.), ABT761 (5-LO inhibitor, AbbottLaboratories), Zyflo® (5-LO inhibitor, Abbott Laboratories), BT061(anti-CD4 mAb, Boehringer Ingelheim GmbH), Corus (inhaled lidocaine todecrease eosinophils, Gilead Sciences Inc), Prograf® (IL-2-mediatedT-cell activation inhibitor, Astellas Pharma), Bimosiamose PFIZER INC(selectin inhibitor, Pfizer Inc), R411 (α4 β1/α4 β7 integrin antagonist,Roche Holdings Ltd), Tilade® (inflammatory mediator inhibitor,Sanofi-Aventis), Orenica® (T-cell co-stimulation inhibitor,Bristol-Myers Squibb Company), Soliris® (anti-CS, AlexionPharmaceuticals Inc), Entorken® (Farmacija d.o.o.), Excellair® (Sykkinase siRNA, ZaBeCor Pharmaceuticals, Baxter International Inc), KB003(anti-GMCSF mAb, KaloBios Pharmaceuticals), Cromolyn sodiums (inhibitrelease of mast cell mediators): Cromolyn sodium BOEHRINGER (BoehringerIngelheim GmbH), Cromolyn sodium TEVA (Teva Pharmaceutical IndustriesLtd), Intal (Sanofi-Aventis), BI1744CL (oldaterol (β2-adrenoceptorantagonist) and tiotropium, Boehringer Ingelheim GmbH), NFκ-Binhibitors, CXR2 antagaonists, FILE inhibitors, HMG-CoA reductaseinhibitors and the like.

Anti-inflammatory agents also include compounds that inhibit/decreasecell signaling by inflammatory molecules like cytokines (e.g., IL-1,IL-4, IL-5, IL-6, IL-9, IL-13, IL-18 IL-25, IFN-α, IFN-β, and others),CC chemokines CCL-1-CCL28 (some of which are also known as, for example,MCP-1, CCL2, RANTES), CXC chemokines CXCL1-CXCL17 (some of which arealso know as, for example, IL-8, MIP-2), growth factors (e.g., GM-CSF,NGF, SCF, TGF-β, EGF, VEGF and others) and/or their respectivereceptors.

Some examples of the aforementioned anti-inflammatoryantagonists/inhibitors include ABN912 (MCP-1/CCL2, Novartis AG), AMG761(CCR4, Amgen Inc), Enbrel® (TNF, Amgen Inc, Wyeth), huMAb OX40LGENENTECH (TNF superfamily, Genentech Inc, AstraZeneca PLC), R4930 (TNFsuperfamily, Roche Holding Ltd), SB683699/Firategrast (VLA4,GlaxoSmithKline PLC), CNT0148 (TNFα, Centocor, Inc, Johnson & Johnson,Schering-Plough Corp); Canakinumab (IL-1β, Novartis); IsrapafantMITSUBISHI (PAF/IL-5, Mitsubishi Tanabe Pharma Corporation); IL-4 andIL-4 receptor antagonists/inhibitors: AMG317 (Amgen Inc), BAY169996(Bayer AG), AER-003 (Aerovance), APG-201 (Apogenix); IL-5 and IL-5receptor antagonists/inhibitors: MEDI563 (AstraZeneca PLC, MedImmune,Inc), Bosatria® (GlaxoSmithKline PLC), Cinquil® (Ception Therapeutic),TMC120B (Mitsubishi Tanabe Pharma Corporation), Bosatria(GlaxoSmithKline PLC), Reslizumab SCHERING (Schering-Plough Corp);MEDI528 (IL-9, AstraZeneca, MedImmune, Inc); IL-13 and IL-13 receptorantagonists/inhibitors: TNX650 GENENTECH (Genentech), CAT-354(AstraZeneca PLC, MedImmune), AMG-317 (Takeda Pharmaceutical CompanyLimited), MK6105 (Merck & Co Inc), IMA-026 (Wyeth), IMA-638 Anrukinzumab(Wyeth), MILR1444A/Lebrikizumab (Genentech), QAX576 (Novartis), CNTO-607(Centocor), MK-6105 (Merck, CSL); Dual IL-4 and IL-13 inhibitors:AIR645/ISIS369645 (ISIS Altair), DOM-0910 (GlaxoSmithKline, Domantis),Pitrakinra/AER001/Aerovant™ (Aerovance Inc), AMG-317 (Amgen), and thelike.

Suitable steroids include corticosteroids, combinations ofcorticosteroids and LABAs, combinations of corticosteroids and LAMAs,combinations of corticosteroids, LABAs and LAMAs, and the like.

Suitable corticosteroids include budesonide, fluticasone, flunisolide,triamcinolone, beclomethasone, mometasone, ciclesonide, dexamethasone,and the like.

Examples of budesonide formulations include Captisol-Enabled® BudesonideSolution for Nebulization (AstraZeneca PLC), Pulmicort® (AstraZenecaPLC), Pulmicort® Flexhaler (AstraZeneca Plc), Pulmicort® HFA-MDI(AstraZeneca PLC), Pulmicort Respules® (AstraZeneca PLC), Inflammide(Boehringer Ingelheim GmbH), Pulmicort® HFA-MDI (SkyePharma PLC), UnitDose Budesonide ASTRAZENECA (AstraZeneca PLC), Budesonide Modulite(Chiesi Farmaceutici S.p.A), CHF5188 (Chiesi Farmaceutici S.p.A),Budesonide ABBOTT LABS (Abbott Laboratories), Budesonide clickhaler(Vestura Group PLC), Miflonide (Novartis AG), Xavin (Teva PharmaceuticalIndustries Ltd.), Budesonide TEVA (Teva Pharmaceutical Industries Ltd.),Symbicort® (AstraZeneca K.K., AstraZeneca PLC), VR632 (Novartis AG,Sandoz International GmbH), and the like.

Examples of fluticasone propionate formulations include FlixotideEvohaler (GlaxoSmithKline PLC), Flixotide Nebules (GlaxoSmithKline Plc),Flovent® (GlaxoSmithKline Plc), Flovent® Diskus (GlaxoSmithKline PLC),Flovent® HFA (GlaxoSmithKline PLC), Flovent® Rotadisk (GlaxoSmithKlinePLC), Advair® HFA (GlaxoSmithKline PLC, Theravance Inc), Advair Diskus®(GlaxoSmithKline PLC, Theravance Inc.), VR315 (Novartis AG, VecturaGroup PLC, Sandoz International GmbH), and the like. Other formulationsof fluticasone include fluticasone as Flusonal (Laboratorios Almirall,S.A.), fluticasone furoate as GW685698 (GlaxoSmithKline PLC, ThervanceInc.), Plusvent (Laboratorios Almirall, S.A.), Flutiform® (AbbottLaboratories, SkyePharma PLC), and the like.

Examples of flunisolide formulations include Aerobid® (ForestLaboratories Inc), Aerospan® (Forest Laboratories Inc), and the like.Examples of triamcinolone include Triamcinolone ABBOTT LABS (AbbottLaboratories), Azmacort® (Abbott Laboratories, Sanofi-Aventis), and thelike. Examples of beclomethasone dipropionate include Beclovent(GlaxoSmithKline PLC), QVAR® (Johnson & Johnson, Schering-Plough Corp,Teva Pharmaceutical Industries Ltd), Asmabec clickhaler (Vectura GroupPLC), Beclomethasone TEVA (Teva Pharmaceutical Industries Ltd), Vanceril(Schering-Plough Corp), BDP Modulite (Chiesi Farmaceutici S.p.A.),Clenil (Chiesi Farmaceutici S.p.A), Beclomethasone dipropionate TEVA(Teva Pharmaceutical Industries Ltd), and the like. Examples ofmometasone include QAB149 Mometasone furoate (Schering-Plough Corp),QMF149 (Novartis AG), Fomoterol fumarate, mometoasone furoate(Schering-Plough Corp), MFF258 (Novartis AG, Merck & Co Inc), Asmanex®Twisthaler (Schering-Plough Corp), and the like. Examples of cirlesonideinclude Alvesco® (Nycomed International Management GmbH, Sepracor,Sanofi-Aventis, Tejin Pharma Limited), Alvesco® Combo (NycomedInternational Management GmbH, Sanofi-Aventis), Alvesco® HFA (NycomedInternational Management GmbH, Sepracor Inc), and the like. Examples ofdexamethasone include DexPak® (Merck), Decadron® (Merck), Adrenocot,CPC-Cort-D, Decaject-10, Solurex and the like. Other corticosteroidsinclude Etiprednol dicloacetate TEVA (Teva Pharmaceutical IndustriesLtd), and the like.

Combinations of corticosteroids and LABAs include salmeterol withfluticasone, formoterol with budesonide, formoterol with fluticasone,formoterol with mometasone, indacaterol with mometasone, and the like.

Examples of salmeterol with fluticasone include Plusvent (LaboratoriosAlmirall, S.A.), Advair® HFA (GlaxoSmithKline PLC), Advair® Diskus(GlaxoSmithKline PLV, Theravance Inc), VR315 (Novartis AG, Vectura GroupPLC, Sandoz International GmbH) and the like. Examples of vilanterolwith fluticasone include GSK642444 with fluticasone and the like.Examples of formoterol with budesonide include Symbicort® (AstraZenecaPLC), VR632 (Novartis AG, Vectura Group PLC), and the like. Examples offormoterol with fluticasone include Flutiform® (Abbott Laboratories,SkyePharma PLC), and the like. Examples of formoterol with mometasoneinclude Dulera®/MFF258 (Novartis AG, Merck & Co Inc), and the like.Examples of indacaterol with mometasone include QAB149 Mometasonefuroate (Schering-Plough Corp), QMF149 (Novartis AG), and the like.Combinations of corticosteroids with LAMAS include fluticasone withtiotropium, budesonide with tiotropium, mometasone with tiotropium,salmeterol with tiotropium, formoterol with tiotropium, indacaterol withtiotropium, vilanterol with tiotropium, and the like. Combinations ofcorticosteroids with LAMAs and LABAs include, for example, fluticasonewith salmeterol and tiotropium.

Other anti-asthma molecules include: ARD111421 (VIP agonist, AstraZenecaPLC), AVE0547 (anti-inflammatory, Sanofi-Aventis), AVE0675 (TLR agonist,Pfizer, Sanofi-Aventis), AVE0950 (Syk inhibitor, Sanofi-Aventis),AVE5883 (NK1/NK2 antagonist, Sanofi-Aventis), AVE8923 (tryptase betainhibitor, Sanofi-Aventis), CGS21680 (adenosine A2A receptor agonist,Novartis AG), ATL844 (A2B receptor antagonist, Novartis AG), BAY443428(tryptase inhibitor, Bayer AG), CHF5407 (M3 receptor inhibitor, ChiesiFarmaceutici S.p.A.), CPLA2 Inhibitor WYETH (CPLA2 inhibitor, Wyeth),IMA-638 (IL-13 antagonist, Wyeth), LAS100977 (LABA, LaboratoriosAlmirall, S.A.), MABA (M3 and (32 receptor antagonist, ChiesiFarmaceutici S.p.A), R1671 (mAb, Roche Holding Ltd), CS003 (Neurokininreceptor antagonist, Daiichi Sankyo Company, Limited), DPC168 (CCRantagonist, Bristol-Myers Squibb), E26 (anti-IgE, Genentech Inc), HAE1(Genentech), IgE inhibitor AMGEN (Amgen Inc), AMG853 (CRTH2 and D2receptor antagonist, Amgen), IPL576092 (LSAID, Sanofi-Aventis), EPI2010(antisense adenosine 1, Chiesi Farmaceutici S.p.A.), CHF5480 (PDE-4inhibitor, Chiesi Farmaceutici S.p.A.), KI04204 (corticosteroid, AbbottLaboratories), SVT47060 (Laboratorios Salvat, S.A.), VML530 (leukotrienesynthesis inhibitor, Abbott Laboratories), LAS35201 (M3 receptorantagonist, Laboratorios Almirall, S.A.), MCC847 (D4 receptorantagonist, Mitsubishi Tanabe Pharma Corporation), MEM 1414 (PDE-4inhibitor, Roche), TA270 (5-LO inhibitor, Chugai Pharmaceutical Co Ltd),TAK661 (eosinophil chemotaxis inhibitor, Takeda Pharmaceutical CompanyLimited), TBC4746 (VLA-4 antagonist, Schering-Plough Corp), VR694(Vectura Group PLC), PLD177 (steroid, Vectura Group PLC), KI03219(corticosteroid+LABA, Abbott Laboratories), AMG009 (Amgen Inc), AMG853(D2 receptor antagonist, Amgen Inc);

AstraZeneca PLC: AZD1744 (CCR3/histamine-1 receptor antagonist, AZD1419(TLR9 agonist), Mast Cell inhibitor ASTRAZENECA, AZD3778 (CCRantagonist), DSP3025 (TLR7 agonist), AZD1981 (CRTh2 receptorantagonist), AZD5985 (CRTh2 antagonist), AZD8075 (CRTh2 antagonist),AZD1678, AZD2098, AZD2392, AZD3825 AZD8848, AZD9215, ZD2138 (5-LOinhibitor), AZD3199 (LABA);

GlaxoSmithKline PLC: GW328267 (adenosine A2 receptor agonist), GW559090(α4 integrin antagonist), GSK679586 (mAb), GSK597901 (adrenergic β2agonist), AM103 (5-LO inhibitor), GSK256006 (PDE4 inhibitor), GW842470(PDE-4 inhibitor), GSK870086 (glucocorticoid agonist), GSK159802 (LABA),GSK256066 (PDE-4 inhibitor), GSK642444 (LABA, adrenergic β2 agonist),GSK64244 and Revolair (fluticasone/vilanterol), GSK799943(corticosteroid), GSK573719 (mAchR antagonist), and GSK573719.

Pfizer Inc: PF3526299, PF3893787, PF4191834 (FLAP antagonist), PF610355(adrenergic β2 agonist), CP664511 (α4β1/VCAM-1 interaction inhibitor),CP609643 (inhibitor of α4β1/VCAM-1 interactions), CP690550 (JAK3inhibitor), SAR21609 (TLR9 agonist), AVE7279 (Th1 switching), TBC4746(VLA-4 antagonist); R343 (IgE receptor signaling inhibitor), SEP42960(adenosine A3 antagonist);

Sanofi-Aventis: MLN6095 (CrTH2 inhibitor), SAR137272 (A3 antagonist),SAR21609 (TLR9 agonist), SAR389644 (DPI receptor antagonist), SAR398171(CRTH2 antagonist), SSR161421 (adenosine A3 receptor antagonist);

Merck & Co Inc: MK0633, MK0633, MK0591 (5-LO inhibitor), MK886(leukotriene inhibitor), BI01211 (VLA-4 antagonist); Novartis AG: QAE397(long-acting corticosteroid), QAK423, QAN747, QAP642 (CCR3 antagonist),QAX935 (TLR9 agonist), NVA237 (LAMA).

Suitable expectorants include guaifenesin, guaiacolculfonate, ammoniumchloride, potassium iodide, tyloxapol, antimony pentasulfide and thelike.

Suitable vaccines include nasally inhaled influenza vaccines and thelike.

Suitable macromolecules include proteins and large peptides,polysaccharides and oligosaccharides, and DNA and RNA nucleic acidmolecules and their analogs having therapeutic, prophylactic ordiagnostic activities. Proteins can include antibodies such asmonoclonal antibodies. Nucleic acid molecules include genes, antisensemolecules such as siRNAs that bind to complementary DNA, RNAi, shRNA,microRNA, RNA, or ribosomes to inhibit transcription or translation.Preferred macromolecules have a molecular weight of at least 800 Da, atleast 3000 Da or at least 5000 Da.

Selected macromolecule drugs for systemic applications: Ventavis®(Iloprost), Calcitonin, Erythropoietin (EPO), Factor IX, GranulocyteColony Stimulating Factor (G-CSF), Granulocyte Macrophage Colony,Stimulating Factor (GM-CSF), Growth Hormone, Insulin, Interferon Alpha,Interferon Beta, Interferon Gamma, Luteinizing Hormone Releasing Hormone(LHRH), follicle stimulating hormone (FSII), Ciliary NeurotrophicFactor, Growth Hormone Releasing Factor (GRF), Insulin-Like GrowthFactor, Insulinotropin, Interleukin-1 Receptor Antagonist,Interleukin-3, Interleukin-4, Interleukin-6, Macrophage ColonyStimulating Factor (M-CSF), Thymosin Alpha 1, IIb/IIIa Inhibitor,Alpha-1 Antitrypsin, Anti-RSV Antibody, palivizumab, motavizumab, andALN-RSV, Cystic Fibrosis Transmembrane Regulator (CFTR) Gene,Deoxyribonuclase (DNase), Heparin, Bactericidal/Permeability IncreasingProtein (BPI), Anti-Cytomegalovirus (CMV) Antibody, Interleukin-1Receptor Antagonist, and the like. GLP-1 analogs (liraglutide,exenatide, etc.), Domain antibodies (dAbs), Pramlintide acetate(Symlin), Leptin analogs, Synagis (palivizumab, MedImmune) andcisplatin.

Selected therapeutics helpful for chronic maintenance of CF includeantibiotics/macrolide antibiotics, bronchodilators, inhaled LABAs, andagents to promote airway secretion clearance. Suitable examples ofantibiotics/macrolide antibiotics include tobramycin, azithromycin,ciprofloxacin, colistin, aztreonam and the like. Another exemplaryantibiotic/macrolide is levofloxacin. Suitable examples ofbronchodilators include inhaled short-acting beta₂ agonists such asalbuterol, and the like. Suitable examples of inhaled LABAs includesalmeterol, formoterol, and the like. Suitable examples of agents topromote airway secretion clearance include Pulmozyme (dornase alfa,Genentech), hypertonic saline, DNase, heparin and the like. Selectedtherapeutics helpful for the prevention and/or treatment of CF includeVX-770 (Vertex Pharmaceuticals) and amiloride.

Selected therapeutics helpful for the treatment of idiopathic pulmonaryfibrosis include Metelimumab (CAT-192) (TGF-β1 mAb inhibitor, Genzyme),Aerovant™ (AER001, pitrakinra) (Dual IL-13, IL-4 protein antagonist,Aerovance), Aeroderm™ (PEGylated Aerovant, Aerovance), microRNA, RNAi,and the like.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises an antibiotic, such as telavancin,tuberculosis-mycobacterium antibiotics, tobramycin, azithromycin,ciprofloxacin, colistin, and the like. In a further preferredembodiment, the respirable dry powder or respirable dry particlecomprises levofloxacin. In a further preferred embodiment, therespirable dry powder or respirable dry particle comprises aztreonam ora pharmaceutically acceptable salt thereof (i.e., Cayston®). In afurther preferred embodiment, the respirable dry powder or respirabledry particle does not comprise tobramycin. In a further preferredembodiment, the respirable dry powder or respirable dry particle doesnot comprise levofloxacin. In a further preferred embodiment, therespirable dry powder or respirable dry particle does not compriseCayston®.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises a LABA, such as salmeterol, formoterol and isomers(e.g. arformoterol), clenbuterol, tulobuterol, vilanterol (Revolair™),indacaterol, carmoterol, isoproterenol, procaterol, bambuterol,milveterol, and the like. In a further preferred embodiment, therespirable dry powder or respirable dry particle comprises formoterol.In a further preferred embodiment, the respirable dry powder orrespirable dry particle comprises salmeterol. When the dry powders areintended for treatment of CF, preferred additional therapeutic agentsare short-acting beta agonists (e.g., albuterol), antibiotics (e.g.,levofloxacin), recombinant human deoxyribonuclease I (e.g., dornasealfa, also known as DNAse), sodium channel blockers (e.g., amiloride),and combinations thereof.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises a LAMA, such as tiotroprium, glycopyrrolate,aclidinium, ipratropium and the like. In a further preferred embodiment,the respirable dry powder or respirable dry particle comprisestiotropium.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises a corticosteroid, such as budesonide, fluticasone,flunisolide, triamcinolone, beclomethasone, mometasone, ciclesonide,dexamethasone, and the like. In a further preferred embodiment, therespirable dry powder or respirable dry particle comprises fluticasone.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises a combination of two or more of the following; aLABA, a LAMA, and a corticosteroid. In a further preferred embodiment,the respirable dry powder or respirable dry particle comprisesfluticasone and salmeterol. In a further preferred embodiment, therespirable dry powder or respirable dry particle comprises fluticasone,salmeterol, and tiotropium.

When an additional therapeutic agent is administered to a patient with adry powder or dry particles disclosed herein, the agent and the drypowder or dry particles are administered to provide substantial overlapof pharmacological activity, and the additional therapeutic agent can beadministered to the patient before, substantially at the same time, orafter the dry powder or dry particles described herein. For example, aLABA such as formoterol, or a short-acting beta agonist such asalbuterol can be administered to the patient before a dry powder or dryparticle, as described herein, is administered.

In preferred embodiments, the respirable dry powder or respirable dryparticle does not comprise a surfactant, such asL-alpha-phosphatidylcholine dipalmitoyl (“DPPC”), diphosphatidylglycerol (DPPG), 1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS),1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC),1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1-palmitoyl-2-oleoylphosphatidylcholine (POPC), fatty alcohols,polyoxyethylene-9-lauryl ether, surface active fatty, acids, sorbitantrioleate (Span 85), glycocholate, surfactin, poloxomers, sorbitan fattyacid esters, tyloxapol, phospholipids, or alkylated sugars.

It is generally preferred that the respirable dry particles and drypowders do not contain salts, excipients, or other active ingredientsthat have a molecular weight of greater than about 1 kilodalton (1000dalton, Da). For example, the respirable particles of the inventionpreferably do not contain a protein, a polypeptide, oligopeptides,nucleic acid or an oligonucleotide with a molecular weight of greaterthan 1 KDa, great than about 900 Da, greater than about 800 Da, greaterthan about 700 Da, or greater than about 600 Da.

Because the respirable dry powders and respirable dry particlesdescribed herein contain salts, they may be hygroscopic. Accordingly itis desirable to store or maintain the respirable dry powders andrespirable dry particles under conditions to prevent hydration of thepowders. For example, if it is desirable to prevent hydration, therelative humidity of the storage environment should be less than 75%,less than 60%, less than 50%, less than 40%, less than 30%, less than25%, less than 20%, less than 15%, less than 10%, or less than 5%humidity. The respirable dry powders and respirable dry particles can bepackaged (e.g., in sealed capsules, blisters, vials) under theseconditions.

The invention also relates to respirable dry powders or respirable dryparticles produced by preparing a feedstock solution, emulsion orsuspension and spray drying the feedstock according to the methodsdescribed herein. The feedstock can be prepared using (a) a calciumsalt, such as calcium lactate or calcium chloride, in an amount of atleast about 25% by weight (e.g., of total solutes used for preparing thefeedstock) and (b) a sodium salt, such as sodium citrate, sodiumchloride or sodium sulfate, in an amount of at least about 1% by weight(e.g., of total solutes used for preparing the feedstock). If desired,one or more excipient, such as leucine can be added to the feedstock inan amount of about 74% or less by weight (e.g., of total solutes usedfor preparing the feedstock). For example, the calcium salt used forpreparing the feedstock can be in an amount of at least about 30%, atleast about 35%, at least about 40%, at least about 50%, at least about60% or at least about 70% by weight of total solutes used for preparingthe feedstock. The sodium salt used for preparing the feedstock, forexample, can be in an amount of at least about 2%, at least about 3%, atleast about 4%, at least about 5%, at least about 6%, at least about 7%,at least about 8%, at least about 9%, at least about 10%, at least about20%, at least about 25%, at least about 30%, at least about 40%, atleast about 50%, at least about 55% or at least about 65% by weight oftotal solutes used for preparing the feedstock. The excipient added tothe feedstock, for example, can be in an amount about 50% or less, about30% or less, about 20% or less, about 10% or less, about 9% or less,about 8% or less, about 7% or less, about 6% or less, about 5% or less,about 4% or less, about 3% or less, about 2% or less, or about 1% orless by weight of total solutes used for preparing the feedstock.Alternatively, the excipient can be about 10% to about 90%, about 10% toabout 50%, about 20% to about 40%, about 50% to about 90%, about 60% toabout 80%, about 40% to about 60% of total solute used for preparing thefeedstock.

In an embodiment, the respirable dry powders or respirable dry particlesof the invention can be obtained by (1) preparing a feedstock comprising(a) a dry solute containing in percent by weight of the total dry soluteabout 10.0% leucine, about 35.1% calcium chloride and about 54.9% sodiumcitrate and (b) one or more suitable solvents for dissolution of thesolute and formation of the feedstock, and (2) spray drying thefeedstock. In another embodiment, the respirable dry powders orrespirable dry particles of the invention can be obtained by (1)preparing a feedstock comprising (a) a dry solute containing in percentby weight of the total dry solute about 10.0% leucine, about 58.6%calcium lactate and about 31.4% sodium chloride and (b) one or moresuitable solvents for dissolution of the solute and formation of thefeedstock, and (2) spray drying the feedstock. In another embodiment,the respirable dry powders or respirable dry particles of the inventioncan be obtained by (1) preparing a feedstock comprising (a) a dry solutecontaining in percent by weight of the total dry solute about 10.0%leucine, about 39.6% calcium chloride and about 50.44% sodium sulfateand (b) one or more suitable solvents for dissolution of the solute andformation of the feedstock and (2) spray drying the feedstock. Inanother embodiment, the respirable dry powders or respirable dryparticles of the invention can be obtained by (1) preparing a feedstockcomprising (a) a dry solute containing in percent by weight of the totaldry solute about 10.0% maltodextrin, about 58.6% calcium lactate andabout 31.4% sodium chloride and (b) one or more suitable solvents fordissolution of the solute and formation of the feedstock, and (2) spraydrying the feedstock. In another embodiment, the respirable dry powdersor respirable dry particles of the invention can be obtained by (1)preparing a feedstock comprising (a) a dry solute containing in percentby weight of the total dry solute about 20.0% leucine, about 75.0%calcium lactate and about 5.0% sodium chloride and (b) one or moresuitable solvents for dissolution of the solute and formation of thefeedstock, and (2) spray drying the feedstock. In another embodiment,the respirable dry powders or respirable dry particles of the inventioncan be obtained by (1) preparing a feedstock comprising (a) a dry solutecontaining in percent by weight of the total dry solute about 37.5%leucine, about 58.6% calcium lactate and about 3.9% sodium chloride andb) one or more suitable solvents for dissolution of the solute andformation of the feedstock, and (2) spray drying the feedstock. Inanother embodiment, the ratio of Ca²⁺ to Na⁺ on a molar basis is about8:1 to about 2:1 or about. As described herein, various methods (e.g.,static mixing, bulk mixing) can be used for mixing the solutes andsolvents to prepare feedstocks, which are known in the art. If desired,other suitable methods of mixing may be used. For example, additionalcomponents that cause or facilitate the mixing can be included in thefeedstock. For example, carbon dioxide produces fizzing or effervescenceand thus can serve to promote physical mixing of the solute andsolvents. Various salts of carbonate or bicarbonate can promote the sameeffect that carbon dioxide produces and, therefore, can be used inpreparation of the feedstocks of the invention. If desired, when a solidcomponent (solute) of the formulation does not fully dissolve in thesolvent or alternatively begins to precipitate out from solution priorto atomization, the resulting suspension can be spray dried.

In preferred embodiments, the respirable dry powders or respirable dryparticles of the invention possess aerosol characteristics that permiteffective delivery of the respirable dry particles to the respiratorysystem without the use of propellants.

In an embodiment, the respirable dry powders or respirable dry particlesof the invention can be produced through an ion exchange reaction. Incertain embodiments of the invention, two saturated or sub-saturatedsolutions are fed into a static mixer in order to obtain a saturated orsupersaturated solution post-static mixing. Preferably, the post-mixedsolution is supersaturated. The two solutions may be aqueous or organic,but are preferably substantially aqueous. The post-static mixingsolution is then fed into the atomizing unit of a spray dryer. In apreferable embodiment, the post-static mixing solution is immediatelyfed into the atomizer unit. Some examples of an atomizer unit include atwo-fluid nozzle, a rotary atomizer, or a pressure nozzle. Preferably,the atomizer unit is a two-fluid nozzle. In one embodiment, thetwo-fluid nozzle is an internally mixing nozzle, meaning that the gasimpinges on the liquid feed before exiting to the most outward orifice.In another embodiment, the two-fluid nozzle is an externally mixingnozzle, meaning that the gas impinges on the liquid feed after exitingthe most outward orifice.

Salts of divalent metal cations (e.g., calcium, magnesium) can beco-formulated with an excipient, and optionally salts of monovalentmetal cations and/or an additional therapeutic agent to form respirabledry particles. Suitable excipients include, for example, sugars (e.g.,lactose, trehalose, maltodextrin), polysaccharides (e.g. dextrin,maltodextrin, dextran, raffinose), sugar alcohols (e.g., mannitol,xylitol, sorbitol), and amino acids (e.g., glycine, alanine, leucine,isoleucine). Other suitable excipients include, for example,dipalmitoylphosphosphatidylcholine (DPPC), diphosphatidyl glycerol(DPPG), 1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS),1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC),1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1-palmitoyl-2-oleoylphosphatidylcholine (POPC), fatty alcohols,polyoxyethylene-9-lauryl ether, surface active fatty, acids, sorbitantrioleate (Span 85), glycocholate, surfactin, poloxomers, sorbitan fattyacid esters, tyloxapol, phospholipids, alkylated sugars, sodiumphosphate, maltodextrin, human serum albumin (e.g., recombinant humanserum albumin), biodegradable polymers (e.g., PLGA), dextran, dextrin,citric acid, sodium citrate, and the like.

Preferably, the excipients are chosen from one or more of the following;sugars (e.g., lactose, trehalose), polysaccharide (e.g. dextrin,maltodextrin, dextran, raffinose), sugar alcohols (e.g., mannitol,xylitol, sorbitol), and amino acids (e.g., glycine, alanine, leucine,isoleucine). More preferably, the excipients are chosen from one or moreof the following: leucine, mannitol, and maltodextrin. In one aspect ofthe invention, the excipient is not a phospholipid, e.g.dipalmitoylphosphosphatidylcholine (DPPC), diphosphatidyl glycerol(DPPG), 1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS),1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC),1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In another aspect of theinvention, the excipient is not a carboxylate acid or its salt form,e.g. citric acid, sodium citrate.

The dry particles of the invention can be blended with anothertherapeutic agent or co-formulated with another therapeutic agent tomaintain the characteristic high dispersibility of the dry particles anddry powders of the invention. Such blended or co-formulated preparationscan provide dry particles that deliver a therapeutic divalent metalcation (e.g., calcium ion) and one or more additional therapeuticagents, or that are carrier particles that deliver one or moretherapeutic agents that are not divalent metal cations, and can beproduced in a variety of ways. For example, respirable dry particles ofthe invention can be blended with an additional therapeutic agent or thecomponents of the dry particles and dry powders described herein can beco-spray dried with an additional therapeutic agent, such as any one orcombination of the additional thereapeutic agents disclosed herein, toproduce a dry powder. Blended dry powders contain particles of the drypowders and dry particles described herein and particles that contain anadditional therapeutic agent. Preferred additional therapeutic agentsare LABAs (e.g., formoterol, salmeterol), short-acting beta agonists(e.g., albuterol), corticosteroids (e.g., fluticasone), LAMAs (e.g.,tiotropium), antibiotics (e.g., levofloxacin, tobramycin), andcombinations thereof. When the dry powders are intended for treatment ofCF, preferred additional therapeutic agents are short-acting betaagonists (e.g., albuterol), antibiotics (e.g., levofloxacin),recombinant human deoxyribonuclease I (e.g., dornase alfa, also known asDNAse), sodium channel blockers (e.g., amiloride), and combinationsthereof.

As described in exemplified herein, dry powders that contain certaindivalent metal cations (e.g., calcium ions) have anti-viral,anti-bacterial and anti-inflammatory activities. These activities areenhanced in dry powders that also contain an monovalent metal cationsalt (e.g. sodium chloride) and in which the ratio of divalent metalcation to monovalent metal cation (mole:mole) fall within certainranges. For example, dry powders that contain a calcium salt (e.g.calcium lactate) and a sodium salt (e.g., sodium chloride) and in whichthe ratio of calcium ion to sodium ion (mole:mole) is about 1:1 to about16:1, or about 1:1 to about 8:1, or about 1:1 to about 4:1, or about 1:1to about 3.9:1, or about 1:1 to about 3.5:1, or about 2:1 to about 8:1,or about 2:1 to about 4:1, or about 2:1 to about 3.9:1, or about 2:1 toabout 3:5, or about 4:1 can have superior activity relative to otherproportions of calcium salts and sodium salts.

Thus, co-formulated dry powders that can be administered to a subject toprovide the benefits of a divalent metal cation (e.g., calcium) andanother therapeutic agent, can comprise respirable dry particles thatinclude a divalent metal cation salt (e.g. a calcium salt), a monovalentmetal cation salt (e.g., a sodium salt), one or more additionaltherapeutic agents, and optionally an excipient. Preferably, the ratioof calcium ion to sodium ion (mole:mole) in such a respirable dryparticle is within one or more of the ranges described above (forexample, the ratio can be about 4:1). This can be accomplished inseveral way, for example, by co-spray drying an additional therapeuticagent with the divalent salt and monovalent salt components, andoptionally all or a portion of the excipient component, if present, ofthe dry powders and dry particles described herein (e.g., any of theparticular formulations described herein). For example, in someembodiments, the dry particle can contain 0% to about 1% excipient.

Respirable dry particles that contain a divalent metal cation salt (e.g.a calcium salt), a monovalent metal cation salt (e.g., a sodium salt),one or more additional therapeutic agents, and optionally an excipient,in which the ratio of divalent metal cation to monovalent metal cationis within one or more of the ranges described herein, can contain anydesired amount of therapeutic agent. It is generally desirable tomaintain a high load of divalent metal cation salt (e.g. a calcium salt)in the respirable dry particles (e.g. at least about 50% (w/w) calciumsalt), however, when high loads of the additional therapeutic agent aredesired, the respirable dry particles can contain lower amounts ofdivalent metal cation salt (e.g., about 10% to about 50%) and asufficient amount of monovalent cation salt to produce the desired ratioof divalent metal cation to monovalent metal cation.

In some embodiments, the respirable dry particles contain a calciumsalt, a sodium salt and an additional therapeutic agent, wherein theadditional therapeutic agent is present in a concentration of about0.01% (w/w) to about 10% (w/w), or about 0.01% (w/w) to about 20% (w/w),or about 0.01% to about 90%, or about 20% (w/w) to about 90% (w/w), orabout 20% (w/w) to about 80% (w/w), or about 20% (w/w) to about 60%(w/w), or about 20% (w/w) to about 50% (w/w), or about 50% (w/w) toabout 90% (w/w), or about 50% (w/w) to about 80% (w/w), or about 60%(w/w) to about 90% (w/w), or about 60% (w/w) to about 80% (w/w), and theratio of calcium ion to sodium ion (mole:mole) is about 1:1 to about16:1, or about 1:1 to about 8:1, or about 1:1 to about 4:1, or about 1:1to about 3.9:1, or about 1:1 to about 3.5:1, or about 2:1 to about 8:1,or about 2:1 to about 4:1, or about 2:1 to about 3.9:1, or about 2:1 toabout 3:5, or about 4:1. The respirable dry particles preferably aresmall (e.g., VMGD at 1.0 bar of 10 μm or less, preferably 5 μm or less)and dispersible (i.e., possessing 1/4 bar and/or 0.5/4 bar ratios of 2.2or less, as described herein). Preferably, the MMAD of the respirabledry particles is from about 0.5 μm to about 10 μm, more preferably fromabout 1 μm to about 5 μm. Preferably, the respirable dry particles arealso calcium dense, and/or have a tap density of about 0.4 g/cc to about1.2 g/cc, preferably between about 0.55 g/cc and about 1.0 g/cc. Thetherapeutic agent in these embodiments are preferably one or more agentsindependently selected from the group consisting of LABAs (e.g.,formoterol, salmeterol), short-acting beta agonists (e.g., albuterol),corticosteroids (e.g., fluticasone), LAMAs (e.g., tiotropium),antibiotics (e.g., levofloxacin), and combinations thereof. When the drypowders are intended for treatment of CF, preferred additionaltherapeutic agents are short-acting beta agonists (e.g., albuterol),antibiotics (e.g., levofloxacin), recombinant human deoxyribonuclease I(e.g., dornase alfa, also known as DNAse), sodium channel blockers(e.g., amiloride), and combinations thereof.

In more particular embodiments, the respirable dry particles contain acalcium salt (e.g. calcium lactate), a sodium salt (e.g., sodiumchloride) and an additional therapeutic agent wherein the additionaltherapeutic agent is an antibiotic (e.g., levofloxacin) that is presentin a concentration of about 20% (w/w) to about 90% (w/w), or about 20%(w/w) to about 80% (w/w), or about 20% (w/w) to about 60% (w/w), orabout 20% (w/w) to about 50% (w/w), or about 50% (w/w) to about 90%(w/w), or about 50% (w/w) to about 80% (w/w), or about 60% (w/w) toabout 90% (w/w), or about 60% (w/w) to about 80% (w/w), and the ratio ofcalcium ion to sodium ion (mole:mole) is about 1:1 to about 16:1, orabout 1:1 to about 8:1, or about 1:1 to about 4:1, or about 1:1 to about3.9:1, or about 1:1 to about 3.5:1, or about 2:1 to about 4:1, or about2:1 to about 3.9:1, or about 2:1 to about 3:5, or about 4:1.

When it is desirable to retain the relative proportions of divalentsalt, monovalent salt and excipient of any of the particular dry powdersand dry particle formulations described herein, the additionaltherapeutic agent can be added to a solution of the components of thedry powder and the resulting solution spray dried to produce dryparticles that contain the additional therapeutic agent. In suchparticles the amount of divalent salt, monovalent salt and excipient inthe dry particles will each be lower than the amounts in the dry powdersor dry particles described herein, due to the addition of the additionaltherapeutic agent. In one example, the formulation can contain up toabout 20% (w/w) additional therapeutic agent, and the amount of each ofdivalent salt, monovalent salt and excipient are reduced proportionally,but the ratio of the amounts (wt %) of divalent salt:monovalentsalt:excipient is the same as in the dry powders or dry particlesdescribed herein. In another example, the formulation can contain up toabout 6% (w/w) additional therapeutic agent. In a further example, theformulation can contain up to about 1% (w/w) additional therapeuticagent.

In exemplary embodiments, the dry particles are based on FormulationVIII and contain up to about 6% (w/w) of one or more additionaltherapeutic agents, about 70% to about 75% (w/w) calcium lactate, about3% to about 5% (w/w) sodium chloride and about 17% to about 20% (w/w)leucine. In other exemplary embodiments, the dry particles are based onFormulation VII and contain up to about 6% (w/w) of one or moreadditional therapeutic agent, about 45.0% to about 58.6% (w/w) calciumlactate, about 1.9% to about 3.9% (w/w) sodium chloride and about 27.5%to about 37.5% (w/w) leucine. In further exemplary embodiments, the dryparticles are based on Formulation VIII and contain up to about 20%(w/w) of one or more additional therapeutic agents, about 60% to about75% (w/w) calcium lactate, about 2% to about 5% (w/w) sodium chlorideand about 15% to about 20% (w/w) leucine. In other exemplaryembodiments, the dry particles are based on Formulation VII and containup to about 20% (w/w) of one or more additional therapeutic agent, about54.6% to about 58.6% (w/w) calcium lactate, about 1.9% to about 3.9%(w/w) sodium chloride and about 34.5% to about 37.5% (w/w) leucine. Whenthe additional therapeutic agent is potent, a small amount may be usedsuch as 0.01% to about 1% (w/w), and the composition of the dryparticles is substantially the same as Formulation VIII or VII. Theadditional therapeutic agent can be any of the additional therapeuticagents described herein. Preferred additional therapeutic agents areLABAs (e.g., formoterol, salmeterol), short-acting beta agonists (e.g.,albuterol), corticosteroids (e.g., fluticasone), LAMAs (e.g.,tiotropium), antibiotics (e.g., levofloxacin, tobramycin), andcombinations thereof. When the dry powders are intended for treatment ofCF, preferred additional therapeutic agents are short-acting betaagonists (e.g., albuterol), antibiotics (e.g., levofloxacin),recombinant human deoxyribonuclease I (e.g., dornase alfa, also known asDNAse), sodium channel blockers (e.g., amiloride), and combinationsthereof.

In dry powders that contain an additional therapeutic agent, all or aportion of the excipient component in the dry powders or dry particlesdescribed herein can be replaced with one or more additional therapeuticagents. This approach is particularly advantageous for additionaltherapeutic agents that require a higher effective dose, e.g., are nothighly potent, and produces dry particles that deliver the beneficialeffects of calcium cation in the respiratory tract and of the beneficialeffects of the additional therapeutic agent(s). In exemplaryembodiments, the dry particles are based on Formulation VIII and containabout 0.01% to about 20% (w/w) of one or more additional therapeuticagent, about 75% (w/w) calcium lactate, about 5% (w/w) sodium chlorideand about 20% (w/w) or less leucine. In other exemplary embodiments, thedry particles are based on Formulation VII and contain about 0.01% toabout 37.5% (w/w) of one or more additional therapeutic agents, about58.6% (w/w) calcium lactate, about 3.9% (w/w) sodium chloride and about37.5% (w/w) or less leucine. The additional therapeutic agent can be anyof the additional therapeutic agents described herein. Preferredadditional therapeutic agent are LABAs (e.g., formoterol, salmeterol),short-acting beta agonists (e.g., albuterol), corticosteroids (e.g.,fluticasone), LAMAs (e.g., tiotropium), antibiotics (e.g., levofloxacin,tobramycin), and combinations thereof. Particular examples of dry powderof this type are disclosed herein as Formulations X-XX.

In one aspect, salts of divalent cations (e.g., calcium, magnesium) canbe co-formulated with a non-calcium active agent, to make small, highlydispersible powders or large, porous particles. Optionally, theseparticles may include a monovalent cationic salt (e.g., sodium,potassium), and also optionally an excipient (e.g., leucine,maltodextrin, mannitol, lactose). The components can be mixed (e.g.,mixed as one solution, static mixed as two solutions) together in orderto produce a single particle after spray drying.

Some respirable dry powders of the invention comprise respirable dryparticles that contain a divalent metal cation or salt thereof that doesnot on its own have a pharmacological effect, or is present in an amountthat does not produce therapeutic efficacy (e.g., a sub-therapeuticamount such as a low % of divalent metal cation salt (e.g., less thanabout 20%, 15%, 10%, 5% or 3% (w/w)). For example, the respirable dryparticles can contain magnesium ion or a magnesium salt such asmagnesium lactate, magnesium sulfate, magnesium citrate, magnesiumcarbonate, magnesium chloride, magnesium phosphate, or any combinationsthereof. Magnesium lactate and magnesium sulfate are preferred.Respirable dry particles of this type can be large and dispersible, butare preferably small and dispersible and dense in mass (e.g., have ahigh tap density or envelope density) as described herein. Suchparticles can be used as carrier particles to deliver other therapeuticagents, for example, by blending with a therapeutic agent or byincorporating a therapeutic agent into the particle (e.g., by co-spraydrying). Preferred therapeutic agents that can be delivered using thesetypes of particles, particularly when co-spray dried with the otherparticle components, are LABAs (e.g., formoterol, salmeterol),short-acting beta agonists (e.g., albuterol), corticosteroids (e.g.,fluticasone), LAMAs (e.g., tiotropium), antibiotics (e.g., levofloxacin,tobramycin), and combinations thereof. When the dry powders are intendedfor treatment of CF, preferred additional therapeutic agents areshort-acting beta agonists (e.g., albuterol), antibiotics (e.g.,levofloxacin), recombinant human deoxyribonuclease I (e.g., dornasealfa, also known as DNAse), sodium channel blockers (e.g., amiloride),and combinations thereof. Additionally, the respirable dry particle mayalso contain an excipient, e.g. a monovalent salt, a sugar, apolysaccharide, a sugar alcohol, an amino acid, and any combinationthereof.

The relative proportions of divalent metal cation or salt thereof,therapeutic agent and any excipients are selected to provide asufficient amount of the therapeutic agent in the dry powder to allow aneffective dose of the therapeutic agent to be conveniently administeredto a subject, for example by inhalation of the dry powder contained inone or two capsules or blisters (e.g., 50 mg capsule, 40 mg capsules).Accordingly, the amount of therapeutic agent can vary from about 0.01%(w/w) for a potent therapeutic agent (or low molecular weighttherapeutic agent) such as tiotropium, to about 90% (w/w) fortherapeutic agents with lower potency (or higher molecular weight) suchas many antibiotics (e.g., levofloxacin). For example, LABAs (e.g.,formoterol, salmeterol), corticosteroids (e.g., fluticasone), and LAMAs(e.g., tiotropium), are generally highly potent and the respirable dryparticle can contain from about 0.01% (w/w) to about 20% (w/w),preferably about 0.01% (w/w) to about 10% (w/w), or about 0.01% (w/w) toabout 5% (w/w) of these therapeutic agents (i.e., alone or in anycombination). Antibiotics are generally less potent and require higherdoses for therapeutic efficacy. Accordingly, the respirable dry particlecan contain from about 10% (w/w) to about 99% (w/w) antibiotic.Preferably, respirable dry particles that contain antibiotic containfrom about 10% (w/w) to about 80% (w/w), about 25% (w/w) to about 80%(w/w), or about 25% (w/w) to about 75% (w/w) antibiotic.

A sufficient amount of one or more divalent metal cation salts andexcipients (e.g., a monovalent salt, a sugar, a polysaccharide, a sugaralcohol, an amino acid, and any combination thereof) are present in suchrespirable dry particles (by % (w/w)) to provide the desired particleproperties (e.g, size, dispersibility, tap density). In general, theamount of divalent metal cation salt in the respirable dry particle issufficient to provide divalent metal cation in an amount of at leastabout 5% (w/w), for example the respirable dry particle can contain fromabout 20% to about 90% (w/w) divalent metal cation salt. The dryparticles may contain about 5% to about 95%, about 5% to about 90%,about 5% to about 85%, about 5% to about 80%, about 5% to about 75%,about 5% to about 70%, about 5% to about 65%, about 5% to about 60%,about 5% to about 55%, about 5% to about 50%, about 5% to about 45%,about 5% to about 40%, about 5% to about 35%, about 5% to about 30%,about 5% to about 25%, about 5% to about 20%, about 5% to about 15%,about 5% to about 10%, or about 5% to about 8% divalent metal cation. Ina preferred aspect, the dry particles contain about 5% to about 20%divalent cation, in a more preferred aspect, the dry particles containabout 5% to about 15% divalent cation. Excipients, are generally presentin the respirable dry particles in an amount of 0% to about 50%,preferably about 10% to about 50%.

Accordingly, in some embodiments the invention is a respirable drypowder that comprise respirable dry particles that contain a magnesiumsalt and a therapeutic agent, and optionally an excipient (e.g, amonovalent metal salt, a sugar, a polysaccharide, a sugar alcohol, anamino acid, and any combination thereof). The respirable dry particlespreferably are small (e.g., VMGD at 1.0 bar of 10 μm or less, preferably5 μm or less) and dispersible (1/4 bar and/or 0.5/4 bar of 2.2 or less,as described herein). Preferably, the MMAD of the respirable dryparticles is from about 0.5 μm to about 10 μm, more preferably fromabout 1 μm to about 5 μm. Preferably, the respirable dry particles arealso dense, and have a tap density of about 0.4 g/cc to about 1.2 g/cc,preferably between about 0.55 g/cc and about 1.0 g/cc. The magnesiumsalt can be magnesium lactate, magnesium sulfate, magnesium citrate,magnesium carbonate, magnesium chloride, magnesium phosphate or anycombination of the forgoing. In preferred embodiments, the magnesiumsalt is magnesium lactate or magnesium sulfate. The therapeutic agent inthese embodiments are preferably one or more agents independentlyselected from the group consisting of LABAs (e.g., formoterol,salmeterol), short-acting beta agonists (e.g., albuterol),corticosteroids (e.g., fluticasone), LAMAs (e.g., tiotropium),antibiotics (e.g., levofloxacin), and combinations thereof. When the drypowders are intended for treatment of CF, preferred additionaltherapeutic agents are short-acting beta agonists (e.g., albuterol),antibiotics (e.g., levofloxacin), recombinant human deoxyribonuclease I(e.g., dornase alfa, also known as DNAse), sodium channel blockers(e.g., amiloride), and combinations thereof.

In more particular embodiments, the respirable dry powder containsrespirable dry particles that contain at least about 5% (w/w) magnesiumion, and 1) about 5% to about 45% excipient, about 20% to about 90%magnesium salt, and about 0.01% to about 20% therapeutic agent; 2) about0.01% to about 30% excipient, about 20% to about 80% magnesium salt, andabout 20% to about 60% therapeutic agent; or 3) about 0.01% to about 20%excipient, about 20% to about 60% magnesium salt, and about 60% to about99% therapeutic agent. The respirable dry particles preferably are small(e.g., VMGD at 1.0 bar of 10 μm or less, preferably 5 μm or less) anddispersible (1/4 bar and/or 0.5/4 bar of 2.2 or less, as describedherein). Preferably, the MMAD of the respirable dry particles is fromabout 0.5 μm to about 10 μm, more preferably from about 1 μm to about 5μm. Preferably, the respirable dry particles are also dense, and have atap density of about 0.4 g/cc to about 1.2 g/cc, preferably betweenabout 0.55 g/cc and about 1.0 g/cc. The magnesium salt can be magnesiumlactate, magnesium sulfate, magnesium citrate, magnesium carbonate,magnesium chloride, magnesium phosphate or any combination of theforgoing. In preferred embodiments, the magnesium salt is magnesiumlactate or magnesium chloride. The therapeutic agent in theseembodiments are preferably one or more agents independently selectedfrom the group consisting of LABAs (e.g., formoterol, salmeterol),short-acting beta agonists (e.g., albuterol), corticosteroids (e.g.,fluticasone), LAMAs (e.g., tiotropium), antibiotics (e.g.,levofloxacin), and combinations thereof. When the dry powders areintended for treatment of CF, preferred additional therapeutic agentsare short-acting beta agonists (e.g., albuterol), antibiotics (e.g.,levofloxacin), recombinant human deoxyribonuclease I (e.g., dornasealfa, also known as DNAse), sodium channel blockers (e.g., amiloride),and combinations thereof.

Alternatively, the particles may be large, e.g. the dry powder has ageometric diameter (VMGD) between 5 microns and 30 microns. Optionally,the particles are large and the tap density may be between 0.01 g/cc and0.4 glee, or between 0.05 Wee and 0.3 g/cc. For small or large VMGDparticles, the MMAD of the dry powder can be between 0.5 and 10 microns,more preferably between 1 and 5 microns.

In another aspect, the dry particles of the invention are large, porous,and are dispersible. The size of the dry particles can be expressed in avariety of ways. The particles may have VMGD between 5 to 30 μm, orbetween 5 and 20 μm, with a tap density of less than 0.5 g/cc,preferably less than 0.4 g/cc.

Methods for Preparing Dry Powders and Dry Particles

The respirable dry particles and dry powders can be prepared using anysuitable method. Many suitable methods for preparing respirable drypowders and particles are conventional in the art, and include singleand double emulsion solvent evaporation, spray drying, milling (e.g.,jet milling), blending, solvent extraction, solvent evaporation, phaseseparation, simple and complex coacervation, interfacial polymerization,suitable methods that involve the use of supercritical carbon dioxide(CO₂), sonocrystallization, nanoparticle aggregate formation, othersuitable methods, and any combination thereof. Respirable dry particlescan be made using methods for making microspheres or microcapsules knownin the art. These methods can be employed under conditions that resultin the formation of respirable dry particles with desired aerodynamicproperties (e.g., aerodynamic diameter and geometric diameter). Ifdesired, respirable dry particles with desired properties, such as sizeand density, can be selected using suitable methods, such as sieving.

The respirable dry particles are preferably spray dried. Suitablespray-drying techniques are described, for example, by K. Masters in“Spray Drying Handbook”, John Wiley & Sons, New York (1984). Generally,during spray-drying, heat from a hot gas such as heated air or nitrogenis used to evaporate a solvent from droplets formed by atomizing acontinuous liquid feed. When hot air is used, the moisture in the air isat least partially removed before its use. When nitrogen is used, thenitrogen gas can be run “dry”, meaning that no additional water vapor iscombined with the gas. If desired the moisture level of the nitrogen orair can be set before the beginning of spray dry run at a fixed valueabove “dry” nitrogen. If desired, the spray drying or other instruments,e.g., jet milling instrument, used to prepare the dry particles caninclude an inline geometric particle sizer that determines a geometricdiameter of the respirable dry particles as they are being produced,and/or an inline aerodynamic particle sizer that determines theaerodynamic diameter of the respirable dry particles as they are beingproduced.

For spray drying, solutions, emulsions or suspensions that contain thecomponents of the dry particles to be produced in a suitable solvent(e.g., aqueous solvent, organic solvent, aqueous-organic mixture oremulsion) are distributed to a drying vessel via an atomization device.For example, a nozzle or a rotary atomizer may be used to distribute thesolution or suspension to the drying vessel. The nozzle can be atwo-fluid nozzle, which is in an internal mixing setup and an externalmixing setup. For example, a rotary atomizer having a 4- or 24-vanedwheel may be used. Examples of suitable spray dryers that can beoutfitted with either a rotary atomizer or a nozzle, include, MobileMinor Spray Dryer or the Model PSD-1, both manufactured by Niro, Inc.(Denmark). Actual spray drying conditions will vary depending, in part,on the composition of the spray drying solution or suspension andmaterial flow rates. The person of ordinary skill will be able todetermine appropriate conditions based on the compositions of thesolution, emulsion or suspension to be spray dried, the desired particleproperties and other factors. In general, the inlet temperature to thespray dryer is about 90° C. to about 300° C., and preferably is about220° C. to about 285° C. Another preferable range is between 130° C. toabout 200° C. The spray dryer outlet temperature will vary dependingupon such factors as the feed temperature and the properties of thematerials being dried. Generally, the outlet temperature is about 50° C.to about 150° C., preferably about 90° C. to about 120° C., or about 98°C. to about 108° C. Another preferable range is between 65° C. to about110° C., more preferably about 75° C. to about 100° C. If desired, therespirable dry particles that are produced can be fractionated byvolumetric size, for example, using a sieve, or fractioned byaerodynamic size, for example, using a cyclone, and/or further separatedaccording to density using techniques known to those of skill in theart.

To prepare the respirable dry particles of the invention, generally, asolution, emulsions or suspension that contains the desired componentsof the dry powder (i.e., a feed stock) is prepared and spray dried undersuitable conditions. Preferably, the dissolved or suspended solidsconcentration in the feed stock is at least about 1 g/L, at least about2 g/L, at least about 5 g/L, at least about 10 g/L, at least about 15g/L, at least about 20 g/L, at least about 30 g/L, at least about 40g/L, at least about 50 g/L, at least about 60 g/L, at least about 70g/L, at least about 80 g/L, at least about 90 g/L, or at least about 100g/L. The feed stock can be provided by preparing a single solution orsuspension by dissolving or suspending suitable components (e.g., salts,excipients, other active ingredients) in a suitable solvent. Thesolvent, emulsion or suspension can be prepared using any suitablemethods, such as bulk mixing of dry and/or liquid components or staticmixing of liquid components to form a combination. For example, ahydrophillic component (e.g., an aqueous solution) and a hydrophobiccomponent (e.g., an organic solution) can be combined using a staticmixer to form a combination. The combination can then be atomized toproduce droplets, which are dried to form respirable dry particles.Preferably, the atomizing step is performed immediately after thecomponents are combined in the static mixer. Alternatively, theatomizing step is performed on a bulk mixed solution.

In one example, respirable dry particles that contain calcium citrate,sodium chloride and leucine are prepared by spray drying. A first phaseis prepared that comprises an aqueous solution of sodium citrate andleucine. A second phase is prepared that comprises calcium chloride inan appropriate solvent. One or both solutions may be separately heatedas needed to assure solubility of their components. The first and secondphases are then combined in a static mixer to form a combination. Thecombination is spray dried to form respirable dry particles.

The feed stock, or components of the feed stock, can be prepared usingany suitable solvent, such as an organic solvent, an aqueous solvent ormixtures thereof. Suitable organic solvents that can be employed includebut are not limited to alcohols such as, for example, ethanol, methanol,propanol, isopropanol, butanols, and others. Other organic solventsinclude but are not limited to perfluorocarbons, dichloromethane,chloroform, ether, ethyl acetate, methyl tert-butyl ether and others.Co-solvents that can be employed include an aqueous solvent and anorganic solvent, such as, but not limited to, the organic solvents asdescribed above. Aqueous solvents include water and buffered solutions.

The feed stock or components of the feed stock can have any desired pH,viscosity or other properties. If desired, a pH buffer can be added tothe solvent or co-solvent or to the formed mixture. Generally, the pH ofthe mixture ranges from about 3 to about 8.

Respirable dry particles and dry powders can be fabricated and thenseparated, for example, by filtration or centrifugation by means of acyclone, to provide a particle sample with a preselected sizedistribution. For example, greater than about 30%, greater than about40%, greater than about 50%, greater than about 60%, greater than about70%, greater than about 80%, or greater than about 90% of the respirabledry particles in a sample can have a diameter within a selected range.The selected range within which a certain percentage of the respirabledry particles fall can be, for example, any of the size ranges describedherein, such as between about 0.1 to about 3 microns VMGD.

The diameter of the respirable dry particles, for example, their VMGD,can be measured using an electrical zone sensing instrument such as aMultisizer Ile, (Coulter Electronic, Luton, Beds, England), or a laserdiffraction instrument such as a HELOS system (Sympatec, Princeton,N.J.) or a Mastersizer system (Malvern, Worcestershire, UK). Otherinstruments for measuring particle geometric diameter are well known inthe art. The diameter of respirable dry particles in a sample will rangedepending upon factors such as particle composition and methods ofsynthesis. The distribution of size of respirable dry particles in asample can be selected to permit optimal deposition within targetedsites within the respiratory system.

Experimentally, aerodynamic diameter can be determined using time offlight (TOF) measurements. For example, an instrument such as theAerosol Particle Sizer (APS) Spectrometer (TSI Inc., Shoreview, Minn.)can be used to measure aerodynamic diameter. The APS measures the timetaken for individual respirable dry particles to pass between two fixedlaser beams.

Aerodynamic diameter also can be experimentally determined directlyusing conventional gravitational settling methods, in which the timerequired for a sample of respirable dry particles to settle a certaindistance is measured. Indirect methods for measuring the mass medianaerodynamic diameter include the Andersen Cascade Impactor and themulti-stage liquid impinger (MSLI) methods. The methods and instrumentsfor measuring particle aerodynamic diameter are well known in the art.

Tap density is an accepted approximate measure of the envelope massdensity characterizing a particle. The envelope mass density of aparticle of a statistically isotropic shape is defined as the mass ofthe particle divided by the minimum sphere envelope volume within whichit can be enclosed. Features which can contribute to low tap densityinclude irregular surface texture, high particle cohesiveness and porousstructure. Tap density can be measured by using instruments known tothose skilled in the art such as the Dual Platform MicroprocessorControlled Tap Density Tester (Vankel, N.C.), a GeoPyc™ instrument(Micrometrics Instrument Corp., Norcross, Ga.), or SOTAX Tap DensityTester model TD2 (SOTAX Corp., Horsham, Pa.). Tap density can bedetermined using the method of USP Bulk Density and Tapped Density,United States Pharmacopia convention, Rockville, Md., 10^(th)Supplement, 4950-4951, 1999.

Fine particle fraction can be used as one way to characterize theaerosol performance of a dispersed powder. Fine particle fractiondescribes the size distribution of airborne respirable dry particles.Gravimetric analysis, using a Cascade impactor, is one method ofmeasuring the size distribution, or fine particle fraction, of airbornerespirable dry particles. The Andersen Cascade Impactor (ACI) is aneight-stage impactor that can separate aerosols into nine distinctfractions based on aerodynamic size. The size cutoffs of each stage aredependent upon the flow rate at which the ACI is operated. The ACI ismade up of multiple stages consisting of a series of nozzles (i.e., ajet plate) and an impaction surface (i.e., an impaction disc). At eachstage an aerosol stream passes through the nozzles and impinges upon thesurface. Respirable dry particles in the aerosol stream with a largeenough inertia will impact upon the plate. Smaller respirable dryparticles that do not have enough inertia to impact on the plate willremain in the aerosol stream and be carried to the next stage. Eachsuccessive stage of the ACI has a higher aerosol velocity in the nozzlesso that smaller respirable dry particles can be collected at eachsuccessive stage.

If desired, a two-stage collapsed ACI can also be used to measure fineparticle fraction. The two-stage collapsed ACI consists of only the toptwo stages 0 and 2 of the eight-stage ACI, as well as the finalcollection filter, and allows for the collection of two separate powderfractions. Specifically, a two-stage collapsed ACI is calibrated so thatthe fraction of powder that is collected on stage two is composed ofrespirable dry particles that have an aerodynamic diameter of less than5.6 microns and greater than 3.4 microns. The fraction of powder passingstage two and depositing on the final collection filter is thus composedof respirable dry particles having an aerodynamic diameter of less than3.4 microns. The airflow at such a calibration is approximately 60L/min. The FPF (<5.6) has been demonstrated to correlate to the fractionof the powder that is able to reach the lungs of the patient, while theFPF (<3.4) has been demonstrated to correlate to the fraction of thepowder that reaches the deep lung of a patient. These correlationsprovide a quantitative indicator that can be used for particleoptimization.

An ACI can be used to approximate the emitted dose, which herein iscalled gravimetric recovered dose and analytical recovered dose.“Gravimetric recovered dose” is defined as the ratio of the powderweighed on all stage filters of the ACI to the nominal dose. “Analyticalrecovered dose” is defined as the ratio of the powder recovered fromrinsing all stages, all stage filters, and the induction port of the ACIto the nominal dose. The FPF_TD (<5.0) is the ratio of the interpolatedamount of powder depositing below 5.0 μm on the ACI to the nominal dose.The FPF_RD (<5.0) is the ratio of the interpolated amount of powderdepositing below 5.0 μm on the ACI to either the gravimetric recovereddose or the analytical recovered dose.

Another way to approximate emitted dose is to determine how much powderleaves its container, e.g. capture or blister, upon actuation of a drypowder inhaler (DPI). This takes into account the percentage leaving thecapsule, but does not take into account any powder depositing on theDPI. The emitted powder mass is the difference in the weight of thecapsule with the dose before inhaler actuation and the weight of thecapsule after inhaler actuation. This measurement can be called thecapsule emitted powder mass (CEPM) or sometimes termed “shot-weight”.

A Multi-Stage Liquid Impinger (MSLI) is another device that can be usedto measure fine particle fraction. The Multi-Stage Liquid Impingeroperates on the same principles as the ACI, although instead of eightstages, MSLI has five. Additionally, each MSLI stage consists of anethanol-wetted glass frit instead of a solid plate. The wetted stage isused to prevent particle bounce and re-entrainment, which can occur whenusing the ACI.

The geometric particle size distribution can be measured for therespirable dry powder after being emitted from a dry powder inhaler(DPI) by use of a laser diffraction instrument such as the MalvernSpraytec. With the inhaler adapter in the closed-bench configuration, anairtight seal is made to the DPI, causing the outlet aerosol to passperpendicularly through the laser beam as an internal flow. In this way,known flow rates can be drawn through the DPI by vacuum pressure toempty the DPI. The resulting geometric particle size distribution of theaerosol is measured by the photodetectors with samples typically takenat 1000 Hz for the duration of the inhalation and the DV50, GSD, FPF<5.0μm measured and averaged over the duration of the inhalation.

The invention also relates to a method for producing a respirable drypowder comprising respirable dry particles that contain calcium citrateor calcium sulfate. The method comprises a) providing a first liquidfeed stock comprising an aqueous solution of calcium chloride, and asecond liquid feed stock comprising an aqueous solution of sodiumsulfate or sodium citrate; b) mixing the first liquid feed stock and thesecond liquid feed stock to produce a mixture in which an anion exchangereaction occurs to produce a saturated or supersaturated solutioncomprising calcium sulfate and sodium chloride, or calcium citrate andsodium chloride; and c) spray drying the saturated or supersaturatedsolution produced in b) to produce respirable dry particles. The firstliquid feed stock and the second liquid feed stock can be batch mixed orpreferably, static mixed. In some embodiments, the resulting mixture isspray dried, and atomized within 60 minutes, within 30 minutes, within15 minutes, within 10 minutes, within 5 minutes, within 4 minutes,within 3 minutes, within 2 minutes, within 1 minute, within 45 seconds,within 30 seconds, within 15 seconds, within 5 seconds of mixing,preferably static mixing.

The invention also relates to a respirable dry powder or respirable dryparticles produced using any of the methods described herein.

The respirable dry particles of the invention can also be characterizedby the chemical stability of the salts or the excipients that therespirable dry particles comprise. The chemical stability of theconstituent salts can affect important characteristics of the respirableparticles including shelf-life, proper storage conditions, acceptableenvironments for administration, biological compatibility, andeffectiveness of the salts. Chemical stability can be assessed usingtechniques well known in the art. One example of a technique that can beused to assess chemical stability is reverse phase high performanceliquid chromatography (RP-HPLC). Respirable dry particles of theinvention include salts that are generally stable over a long period oftime.

If desired, the respirable dry particles and dry powders describedherein can be further processed to increase stability. An importantcharacteristic of pharmaceutical dry powders is whether they are stableat different temperature and humidity conditions. Unstable powders willabsorb moisture from the environment and agglomerate, thus alteringparticle size distribution of the powder.

Excipients, such as maltodextrin, may be used to create more stableparticles and powders. The maltodextrin may act as an amporphous phasestabilizer and inhibit the components from converting from an amorphousto crystalline state. Alternatively, a post-processing step to help theparticles through the crystallization process in a controlled way (e.g.,on the baghouse at elevated humidity) can be employed with the resultantpowder potentially being further processed to restore its dispersibilityif agglomerates formed during the crystallization process, such as bypassing the particles through a cyclone to break apart the agglomerates.Another possible approach is to optimize around process conditions thatlead to manufacturing particles that are more crystalline and thereforemore stable. Another approach is to use different excipients, ordifferent levels of current excipients to attempt to manufacture morestable forms of the salts.

The respirable dry particles and dry powders described herein aresuitable for inhalation therapies. The respirable dry particles may befabricated with the appropriate material, surface roughness, diameterand tap density for localized delivery to selected regions of therespiratory system such as the deep lung or upper or central airways.

In order to relate the dispersion of powder at different inhalation flowrates, volumes, and from inhalers of different resistances, the energyrequired to perform the inhalation maneuver can be calculated.Inhalation energy can be calculated from the equation E=R²Q²V where E isthe inhalation energy in Joules, R is the inhaler resistance inkPa^(1/2)/LPM, Q is the steady flow rate in L/min and V is the inhaledair volume in L.

The respirable dry powders and dry particles described herein arecharacterized by a high emitted dose (e.g., CEPM of at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%) from a dry powderinhaler when a total inhalation energy of less than about 2 Joules orless than about 1 Joule, or less than about 0.8 Joule, or less thanabout 0.5 Joule, or less than about 0.3 Joule is applied to the drypowder inhaler. For example, an emitted dose of at at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% CEPM of FormulationI or Formulation II contained in a unit dose container, containing about50 mg or about 40 mg of the appropriate formulation, in a dry powderinhaler can be achieved when a total inhalation energy of less thanabout 1 Joule (e.g., less than about 0.8 Joule, less than about 0.5Joule, less than about 0.3 Joule) is applied to the dry powder inhaler.An emitted dose of at least about 70% CEPM of respirable dry powdercontained in a unit dose container, containing about 50 mg or about 40mg of the respirable dry powder, in a dry powder inhaler can be achievedwhen a total inhalation energy of less than about 0.28 Joule is appliedto the dry powder inhaler. The dry powder can fill the unit dosecontainer, or the unit dose container can be at least 40% full, at least50% full, at least 60% full, at least 70% full, at least 80% full, or atleast 90% full. The unit dose container can be a capsule (e.g. size 000,00, 0E, 0, 1, 2, 3, and 4, with respective volumetric capacities of 1.37ml, 950 μl, 7704 680 μl, 480 μl, 360 μl, 270 μl, and 200 μl).Alternatively, the unit dose container can be a blister. The blister canbe packaged as a single blister, or as part of a set of blisters, forexample, 7 blisters, 14 blisters, 28 blisters, or 30 blisters.

Healthy adult populations are predicted to be able to achieve inhalationenergies ranging from 2.9 Joules for comfortable inhalations to 22Joules for maximum inhalations by using values of peak inspiratory flowrate (PIFR) measured by Clarke et al. (Journal of Aerosol Med, 6(2), p.99-110, 1993) for the flow rate Q from two inhaler resistances of 0.02and 0.055 kPa1/2/LPM, with a inhalation volume of 2 L based on both FDAguidance documents for dry powder inhalers and on the work of Tiddens etal. (Journal of Aerosol Med, 19(4), p. 456-465, 2006) who found adultsaveraging 2.2 L inhaled volume through a variety of DPIs.

Mild, moderate and severe adult COPD patients are predicted to be ableto achieve maximum inhalation energies of 5.1 to 21 Joules, 5.2 to 19Joules, and 2.3 to 18 Joules respectively. This is again based on usingmeasured PIFR values for the flow rate Q in the equation for inhalationenergy. The PIER achievable for each group is a function of the inhalerresistance that is being inhaled through. The work of Broeders et al.(Eur Respir J, 18, p. 780-783, 2001) was used to predict maximum andminimum achievable PIER through 2 dry powder inhalers of resistances0.021 and 0.032 kPa1/2/LPM for each.

Similarly, adult asthmatic patients are predicted to be able to achievemaximum inhalation energies of 7.4 to 21 Joules based on the sameassumptions as the COPD population and PIFR data from Broeders et al.

Healthy adults and children, COPD patients, asthmatic patients ages 5and above, and CF patients, for example, are capable of providingsufficient inhalation energy to empty and disperse the dry powderformulations of the invention. For example, a 50 mg dose of FormulationI or Formulation II was found to require only 0.28 Joules to empty morethan 70% of the fill weight in a single inhalation. All the adultpatient populations listed above were calculated to be able to achievegreater than 2 Joules, 7 times more than the inhalational energyrequired. For example, a 25 mg dose of Formulation II was found torequire only 0.16 Joules to empty 80% of the fill weight in a singleinhalation well deagglomerated as illustrated by a Dv50 within 1micrometer of that at much higher inhalation energies. All the adultpatient populations listed above were calculated to be able to achievegreater than 2 Joules, more than an order of magnitude more inhalationalenergy than required.

An advantage of the invention is the production of powders that dispersewell across a wide range of flowrates and are relatively flowrateindependent. The dry particles and powders of the invention enable theuse of a simple, passive DPI for a wide patient population.

Methods

The respirable dry powders and respirable dry particles of the presentinvention are for administration to the respiratory tract. The drypowders and dry particles of the invention can be administered to asubject in need thereof for the treatment of respiratory (e.g.,pulmonary) diseases, such as asthma, airway hyperresponsiveness,seasonal allergic allergy, brochiectasis, chronic bronchitis, emphysema,chronic obstructive pulmonary disease, cystic fibrosis, pulmonaryparenchyal inflammatory conditions and the like, and for the treatmentand/or prevention of acute exacerbations of these chronic diseases, suchas exacerbations caused by viral infections (e.g., influenza virus,parainfluenza virus, respiratory syncytial virus, rhinovirus,adenovirus, metapneumovirus, coxsackie virus, echo virus, corona virus,herpes virus, cytomegalovirus, and the like), bacterial infections(e.g., Streptococcus pneumoniae, which is commonly referred to aspneumococcus, Staphylococcus aureus, Burkholderis ssp., Streptococcusagalactiae, Haemophilus influenzae, Haemophilus parainfluenzae,Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa,Moraxella catarrhalis, Chlamydophila pneumoniae, Mycoplasma pneumoniae,Legionella pneumophila, Serratia marcescens, Mycobacterium tuberculosis,Bordetella pertussis, and the like), fungal infections (e.g.,Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis jiroveci,Coccidioides immitis, and the like) or parasitic infections (e.g.,Toxoplasma gondii, Strongyloides stercoralis, and the like), orenvironmental allergens and irritants (e.g., aeroallergens, includingpollen and cat dander, airborne particulates, and the like).

The dry powders and dry particles of the invention can be administeredto a subject in need thereof for the treatment and/or prevention and/orreducing contagion of infectious diseases of the respiratory tract, suchas pneumonia (including community-acquired pneumonia, nosocomialpneumonia (hospital-acquired pneumonia, HAP: health-care associatedpneumonia, HCAP), ventilator-associated pneumonia (VAP)),ventilator-associated tracheobronchitis (VAT), bronchitis, croup (e.g.,postintubation croup, and infectious croup), tuberculosis, influenza,common cold, and viral infections (e.g., influenza virus, parainfluenzavirus, respiratory syncytial virus, rhinovirus, adenovirus,metapneumovirus, coxsackie virus, echo virus, corona virus, herpesvirus, cytomegalovirus, and the like), bacterial infections (e.g.,Streptococcus pneumoniae, which is commonly referred to as pneumococcus,Staphylococcus aureus, Streptococcus agalactiae, Haemophilus influenzae,Haemophilus parainfluenzae, Klebsiella pneumoniae, Escherichia coli,Pseudomonas aeruginosa, Moraxella catarrhalis, Chlamydophila pneumoniae,Mycoplasma pneumoniae, Legionella pneumophila, Serratia marcescens,Mycobacterium tuberculosis, Bordetella pertussis, and the like), fungalinfections (e.g., Histoplasma capsulatum, Cryptococcus neoformans,Pneumocystis jiroveci, Coccidioides immitis, and the like) or parasiticinfections (e.g., Toxoplasma gondii, Strongyloides stercoralis, and thelike), or environmental allergens and irritants (e.g., aeroallergens,airborne particulates, and the like). Similarly, the respirable dryparticles or dry powders can be administered to a subject in needthereof to prevent or treat chronic infections like bacterialcolonization and biofilm formation that are often seen in those withchronic respiratory diseases like cystic fibrosis and chronicobstructive pulmonary disease. Without wishing to be bound by particulartheory, it is believed that the respirable dry particles or dry powdersdescribed herein may activate cation-regulated ion channels like, forexample, TRP channels (e.g., TRPV, TRPC, TRPM, TRPA channels) andmediate the eventual induction of anti-microbial defenses like, forexample, the secrection of anti-microbial peptides (e.g., alpha-, beta-,theta-defensins), thereby preventing and/or treating microbialinfections.

The respirable dry particles and dry powders can be administered toalter the biophysical and/or biological properties of the mucosal liningof the respiratory tract (e.g, the airway lining fluid) and underlyingtissue (e.g., respiratory tract epithelium). These properties include,for example, gelation at the mucus surface, surface tension of themucosal lining, surface elasticity and/or viscosity of the mucosallining, bulk elasticity and/or viscosity of the mucosal lining. Withoutwishing to be bound by a particular theory, it is believed that thebenefits produced by the respirable dry particles or dry powder and themethods described herein (e.g., therapeutic and prophylactic benefits),result from an increase in the amount of calcium cation (Ca²⁺ providedby the calcium salts in the respirable dry particles or dry powder) inthe respiratory tract (e.g., lung mucus or airway lining fluid) afteradministration of the respirable dry particles or dry powder.

The respirable dry powders and dry particles can be administered toincrease the rate of mucociliary clearance. Clearance of microbes andinhaled particles is an important function of airways to preventrespiratory infection and exposure to or systemic absorption ofpotentially noxious agents. This is performed as an integrated functionby epithelial, mucus-secreting, and immunologic response cells presentat the airway surface. It prominently includes the cilia at theepithelial cell airway surface, whose function is to beat synchronouslyto transport the overlying liquid mucus blanket proximally (toward themouth), where it exits the airway and is swallowed or expectorated.

The respirable dry powders and dry particles can be administered toassist in all of these functions. By increasing surface viscoelasticity,the respirable dry powders and dry particles retain microbes andparticulates at the surface of the airway mucus blanket, where they donot gain systemic exposure to the host. Dry powders and dry particlesinduce water/liquid transport out of the airway epithelial cells, makingthe peri-ciliary liquid layer less viscous and rendering ciliary beatingmore effective in moving and clearing the overlying mucus blanket. Dryparticles and dry powders that contain calcium salts as thepharmacologically active agent, also cause an increase in both ciliarybeat frequency and the force or vigor of ciliary contractions, withresultant increase in clearance velocity of the overlying mucus stream.

Mucociliary clearance is measured by a well-established technique thatmeasures the function and speed of clearance quantitatively using safe,inhaled radioisotope preparation (e.g., Technitium (^(99m)Tc)) insolution. The radioisotope is measured quantitatively by externalscintigraphy. Serial measurements over minutes to several hours allowfor the assessment of velocity of clearance and effect of a drug vs.baseline/control value.

In some aspects, the invention is a method for treating a pulmonarydisease, such as asthma, airway hyperresponsiveness, seasonal allergicallergy, bronchiectasis, chronic bronchitis, emphysema, chronicobstructive pulmonary disease, cystic fibrosis and the like, comprisingadministering to the respiratory tract of a subject in need thereof aneffective amount of respirable dry particles or dry powder, as describedherein.

In other aspects, the invention is a method for treating and/or reducingthe severity of pulmonary parenchyal inflammatory/fibrotic conditions,such as idiopathic pulmonary fibrosis, pulmonary interstitialinflammatory conditions (e.g., sarcoidosis, allergic interstitialpneumonitis (e.g., Farmer's Lung)), fibrogenic dust interstitialdiseases (e.g., asbestosis, silicosis, beryliosis), eosinophilicgranulomatosis/histiocytosis X, collagen vascular diseases (e.g.,rheumatoid arthritis, scleroderma, lupus), Wegner's granulomatosis, andthe like, comprising administering to the respiratory tract of a subjectin need thereof an effective amount of respirable dry particles or drypowder, as described herein.

In other aspects, the invention is a method for the treatment orprevention of acute exacerbations of a chronic pulmonary disease, suchas asthma, airway hyperresponsiveness, seasonal allergic allergy,bronchiectasis, chronic bronchitis, emphysema, chronic obstructivepulmonary disease, cystic fibrosis and the like, comprisingadministering to the respiratory tract of a subject in need thereof aneffective amount of respirable dry particles or dry powder, as describedherein.

In other aspects, the invention is a method for treating, preventingand/or reducing contagion of an infectious disease of the respiratorytract, comprising administering to the respiratory tract of a subject inneed thereof an effective amount of respirable dry particles or drypowder, as described herein.

In still other aspects, the invention is a method for reducinginflammation comprising administering to the respiratory tract of asubject in need thereof an effective amount of respirable dry particlesor dry powders as described herein. Thus, the respirable dry particlesand dry powders can be used to broadly prevent or treat acute and/orchronic inflammation and, in particular, inflammation that characterizesa number of pulmonary diseases and conditions including, asthma, airwayhyperresponsiveness, seasonal allergic allergy, bronchiectasis, chronicbronchitis, emphysema, chronic obstructive pulmonary disease (COPD),cystic fibrosis (CF), pulmonary parenchyal inflammatorydiseases/conditions, and the like. The dry particles and dry powders canbe administered to prevent or treat both the inflammation inherent indiseases like asthma, COPD and CF and the increased inflammation causedby acute exacerbations of those diseases, both of which play a primaryrole in the pathogenesis of the diseases.

In certain particular embodiments of the methods described herein, therespirable dry powders or dry particles described herein areadministered to a patient who has been pretreated with a bronchodilator,or is administered concurrently with a bronchodilator. When the patientis pretreated with a bronchodilator it is preferred that the respirabledry powder or dry particle is administered at a time after thebronchodilator when the onset of bronchodilatory effect is evident or,more preferably, maximal. For example, a short acting beta₂ agonist suchas albuterol, can be administered about 10 minutes to about 30 minutes,preferably, about 15 minutes, prior to administration of the respirabledry powder or dry particles. Pretreatment with a short acting beta₂agonist such as albuterol is particularly preferred for CF patients.Some patients may already be taking bronchodilators, such as LABAs (e.g,fomoterol). Patients with COPD frequently take LABAs to manage theirdisease. Patients that are taking LABAs already receive some degree ofbronchorelaxation due to the effects of the LABAs, and therefore furtherbronchodilation (e.g., using a short acting beta₂ agonist) may not berequired or desired. For these types of patients, respirable dry powderor dry particles can be administered at substantially the same time orconcurrently with the LABA, for example, in a single formulation.

The respirable dry particles and dry powders can be administered to therespiratory tract of a subject in need thereof using any suitablemethod, such as instillation techniques, and/or an inhalation device,such as a dry powder inhaler (DPI) or metered dose inhaler (MDI). Anumber of DPIs are available, such as, the inhalers disclosed is U.S.Pat. Nos. 4,995,385 and 4,069,819, Spinhaler® (Fisons, Loughborough,U.K.), Rotahalers®, Diskhaler® and Diskus® (GlaxoSmithKline, ResearchTriangle Technology Park, North Carolina), FlowCapss® (Hovione, Loures,Portugal), Inhalators® (Boehringer-Ingelheim, Germany), Aerolizer®(Novartis, Switzerland), and others known to those skilled in the art.

Generally, inhalation devices (e.g., DPIs) are able to deliver a maximumamount of dry powder or dry particles in a single inhalation, which isrelated to the capacity of the blisters, capsules (e.g. size 000, 00,0E, 0, 1, 2, 3, and 4, with respective volumetric capacities of 1.37 ml,950 μl, 770 μl, 680 μl, 480 μl, 360 μl, 270 μl, and 200 μl) or othermeans that contain the dry particles or dry powders within the inhaler.Accordingly, delivery of a desired dose or effective amount may requiretwo or more inhalations. Preferably, each dose that is administered to asubject in need thereof contains an effective amount of respirable dryparticles or dry powder and is administered using no more than about 4inhalations. For example, each dose of respirable dry particles or drypowder can be administered in a single inhalation or 2, 3, or 4inhalations. The respirable dry particles and dry powders, arepreferably administered in a single, breath-activated step using abreath-activated DPI. When this type of device is used, the energy ofthe subject's inhalation both disperses the respirable dry particles anddraws them into the respiratory tract.

The respirable dry particles or dry powders can be delivered byinhalation to a desired area within the respiratory tract, as desired.It is well-known that particles with an aerodynamic diameter of about 1micron to about 3 microns, can be delivered to the deep lung. Largeraerodynamic diameters, for example, from about 3 microns to about 5microns can be delivered to the central and upper airways.

In certain embodiments, a dry powder formulation is administered to thesmall airways. In these embodiments, the dry powder preferably containsrespirable particles that have a VMDG and/or MMAD that is suitable fordelivery to the small airways, such as a VMGD and/or MMAD of about 0.5μm to about 3 μm, about 0.75 μm to about 2 μm, or about 1 μm to about1.5 μm.

It is believed that when some dry powders that contain divalent metalsalts as active ingredients are administered, there is a possibilitythat at least some of the respirable dry powder will deposit in the oralcavity and produce an unpleasant “salty mouth” sensation. It isenvisioned that this sensation could lead patients to not comply withtherapeutic instructions or to discontinue therapy. An advantage of therespirable dry powders of this invention is that they are small andhighly dispersible, and therefore, deposition in the oral cavity isreduced and the occurrence of an unpleasant salty mouth sensation isreduced or prevented.

For dry powder inhalers, oral cavity deposition is dominated by inertialimpaction and so characterized by the aerosol's Stokes number (DeHaan etal. Journal of Aerosol Science, 35 (3), 309-331, 2003). For equivalentinhaler geometry, breathing pattern and oral cavity geometry, the Stokesnumber, and so the oral cavity deposition, is primarily affected by theaerodynamic size of the inhaled powder. Hence, factors which contributeto oral deposition of a powder include the size distribution of theindividual particles and the dispersibility of the powder. If the MMADof the individual particles is too large, e.g. above 5 μm, then anincreasing percentage of powder will deposit in the oral cavity.Likewise, if a powder has poor dispersibility, it is an indication thatthe particles will leave the dry powder inhaler and enter the oralcavity as agglomerates. Agglomerated powder will perform aerodynamicallylike an individual particle as large as the agglomerate, therefore evenif the individual particles are small (e.g., MMAD of 5 microns or less),the size distribution of the inhaled powder may have an MMAD of greaterthan 5 μm, leading to enhanced oral cavity deposition.

Therefore, it is desirable to have a powder in which the particles aresmall (e.g., MMAD of 5 microns or less, e.g. between 1 to 5 microns),and are highly dispersible (e.g. 1/4 bar or alternatively, 0.5/4 bar of2.0, and preferably less than 1.5). More preferably, the respirable drypowder is comprised of respirable dry particles with an MMAD between 1to 4 microns or 1 to 3 microns, and have a 1/4 bar less than 1.4, orless than 1.3, and more preferably less than 1.2.

The absolute geometric diameter of the particles measured at 1 bar usingthe HELOS system is not critical provided that the particle's envelopedensity is sufficient such that the MMAD is in one of the ranges listedabove, wherein MMAD is VMGD times the square root of the envelopedensity (MMAD=VMGD*sqrt(envelope density)). If it is desired to delivera high unit dose of salt using a fixed volume dosing container, then,particles of higher envelope density are desired. High envelope densityallows for more mass of powder to be contained within the fixed volumedosing container. Preferable envelope densities are greater than 0.1g/cc, greater than 0.25 g/cc, greater than 0.4 g/cc, greater than 0.5g/cc, and greater than 0.6 g/cc.

The respirable dry powders and particles of the invention can beemployed in compositions suitable for drug delivery via the respiratorysystem. For example, such compositions can include blends of therespirable dry particles of the invention and one or more other dryparticles or powders, such as dry particles or powders that containanother active agent, or that consist of or consist essentially of oneor more pharmaceutically acceptable excipients.

Respirable dry powders and dry particles suitable for use in the methodsof the invention can travel through the upper airways (i.e., theoropharynx and larynx), the lower airways, which include the tracheafollowed by bifurcations into the bronchi and bronchioli, and throughthe terminal bronchioli which in turn divide into respiratory bronchiolileading then to the ultimate respiratory zone, the alveoli or the deeplung. In one embodiment of the invention, most of the mass of respirabledry powders or particles deposit in the deep lung. In another embodimentof the invention, delivery is primarily to the central airways. Inanother embodiment, delivery is to the upper airways.

The respirable dry particles or dry powders of the invention can bedelivered by inhalation at various parts of the breathing cycle (e.g.,laminar flow at mid-breath). An advantage of the high dispersibility ofthe dry powders and dry particles of the invention is the ability totarget deposition in the respiratory tract. For example, breathcontrolled delivery of nebulized solutions is a recent development inliquid aerosol delivery (Dalby et al. in Inhalation Aerosols, edited byHickey 2007, p. 437). In this case, nebulized droplets are released onlyduring certain portions of the breathing cycle. For deep lung delivery,droplets are released in the beginning of the inhalation cycle, whilefor central airway deposition, they are released later in theinhalation.

The highly dispersible powders of this invention provide advantages fortargeting the timing of drug delivery in the breathing cycle and alsolocation in the human lung. Because the respirable dry powders of theinvention can be dispersed rapidly, such as within a fraction of atypical inhalation maneuver, the timing of the powder dispersal can becontrolled to deliver an aerosol at specific times within theinhalation.

With a highly dispersible powder, the complete dose of aerosol can bedispersed at the beginning portion of the inhalation. While thepatient's inhalation flow rate ramps up to the peak inspiratory flowrate, a highly dispersible powder will begin to disperse already at thebeginning of the ramp up and could completely disperse a dose in thefirst portion of the inhalation. Since the air that is inhaled at thebeginning of the inhalation will ventilate deepest into the lungs,dispersing the most aerosol into the first part of the inhalation ispreferable for deep lung deposition. Similarly, for central deposition,dispersing the aerosol at a high concentration into the air which willventilate the central airways can be achieved by rapid dispersion of thedose near the mid to end of the inhalation. This can be accomplished bya number of mechanical and other means such as a switch operated bytime, pressure or flow rate which diverts the patient's inhaled air tothe powder to be dispersed only after the switch conditions are met.

Aerosol dosage, formulations and delivery systems may be selected for aparticular therapeutic application, as described, for example, in Gonda,I. “Aerosols for delivery of therapeutic and diagnostic agents to therespiratory tract,” in Critical Reviews in Therapeutic Drug CarrierSystems, 6: 273-313 (1990); and in Moren, “Aerosol Dosage Forms andFormulations,” in Aerosols in Medicine, Principles, Diagnosis andTherapy, Moren, et al., Eds., Esevier, Amsterdam (1985).

As described herein, it is believed that the therapeutic andprophylactic effects of the respirable dry particles and dry powders arethe result of an increased amount of calcium in the respiratory tract(e.g., lung) following administration of respirable dry particles anddry powders. Accordingly, since the amount of calcium provided can varydepending upon the particular salt selected, dosing can be based on thedesired amount of calcium to be delivered to the lung. For example, onemole of calcium chloride (CaCl₂) dissociates to provide one mole ofCa²⁺, but one mole of calcium citrate can provide three moles of Ca²⁺.

Generally, an effective amount of a pharmaceutical formulation willdeliver a dose of about 0.001 mg Ca²⁺/kg body weight/dose to about 2 mgCa²⁺/kg body weight/dose, about 0.002 mg Ca²⁺/kg body weight/dose toabout 2 mg Ca²⁺/kg body weight/dose, about 0.005 mg Ca²⁺/kg bodyweight/dose to about 2 mg Ca²⁺/kg body weight/dose, about 0.01 mgCa²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg body weight/dose, about0.01 mg Ca²⁺/kg body weight/dose to about 60 mg Ca²⁺/kg bodyweight/dose, about 0.01 mg Ca²⁺/kg body weight/dose to about 50 mg Ca²⁺1kg body weight/dose, about 0.01 mg Ca²⁺/kg body weight/dose to about 40mg Ca²⁺/kg body weight/dose, about 0.01 mg Ca²⁺/kg body weight/dose toabout 30 mg Ca²⁺/kg body weight/dose, about 0.01 mg Ca²⁺/kg bodyweight/dose to about 20 mg Ca²⁺/kg body weight/dose, about 0.01 mgCa²⁺/kg body weight/dose to about 10 mg Ca²⁺/kg body weight/dose, about0.01 mg Ca²⁺/kg body weight/dose to about 5 mg Ca²⁺/kg body weight/dose,about 0.01 mg Ca²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg bodyweight/dose, about 0.02 mg Ca²⁺/kg body weight/dose to about 2 mgCa²⁺/kg body weight/dose, about 0.03 mg Ca²⁺/kg body weight/dose toabout 2 mg Ca²⁺/kg body weight/dose, about 0.04 mg Ca²⁺/kg bodyweight/dose to about 2 mg Ca²⁺/kg body weight/dose, about 0.05 mgCa²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg body weight/dose, about0.1 mg Ca²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg body weight/dose,about 0.1 mg Ca²⁺/kg body weight/dose to about 1 mg Ca²⁺/kg bodyweight/dose, about 0.1 mg Ca²⁺/kg body weight/dose to about 0.5 mgCa²⁺/kg body weight/dose, about 0.2 mg Ca²⁺/kg body weight/dose to about0.5 mg Ca²⁺/kg body weight/dose, about 0.18 mg Ca²⁺/kg body weight/dose,about 0.001 mg Ca²⁺/kg body weight/dose, about 0.005 mg Ca²⁺/kg bodyweight/dose, about 0.01 mg Ca²⁺/kg body weight/dose, about 0.02 mgCa²⁺/kg body weight/dose, or about 0.5 mg Ca²⁺/kg body weight/dose.

In some embodiments the amount of calcium delivered to the respiratorytract (e.g., lungs, respiratory airway) is about 0.001 mg Ca²⁺/kg bodyweight/dose to about 2 mg Ca²⁺/kg body weight/dose, about 0.002 mgCa²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg body weight/dose, about0.005 mg Ca²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg bodyweight/dose, about 0.01 mg Ca²⁺/kg body weight/dose to about 2 mgCa²⁺/kg body weight/dose, about 0.01 mg Ca²⁺/kg body weight/dose toabout 60 mg Ca²⁺/kg body weight/dose, about 0.01 mg Ca²⁺/kg bodyweight/dose to about 50 mg Ca²⁺/kg body weight/dose, about 0.01 mgCa²⁺/kg body weight/dose to about 40 mg Ca²⁺/kg body weight/dose, about0.01 mg Ca²⁺/kg body weight/dose to about 30 mg Ca²⁺/kg bodyweight/dose, about 0.01 mg Ca²⁺/kg body weight/dose to about 20 mgCa²⁺/kg body weight/dose, about 0.01 mg Ca²⁺/kg body weight/dose toabout 10 mg Ca²⁺/kg body weight/dose, about 0.01 mg Ca²⁺/kg bodyweight/dose to about 5 mg Ca²⁺/kg body weight/dose, about 0.01 mgCa²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg body weight/dose, about0.02 mg Ca²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg body weight/dose,about 0.03 mg Ca²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg bodyweight/dose, about 0.04 mg Ca²⁺/kg body weight/dose to about 2 mgCa²⁺/kg body weight/dose, about 0.05 mg Ca²⁺/kg body weight/dose toabout 2 mg Ca²⁺/kg body weight/dose, about 0.1 mg Ca²⁺/kg bodyweight/dose to about 2 mg Ca²⁺/kg body weight/dose, about 0.1 mg Ca²⁺/kgbody weight/dose to about 1 mg Ca²⁺/kg body weight/dose, about 0.1 mgCa²⁺/kg body weight/dose to about 0.5 mg Ca²⁺/kg body weight/dose, about0.2 mg Ca²⁺/kg body weight/dose to about 0.5 mg Ca²⁺/kg bodyweight/dose, about 0.18 mg Ca²⁺/kg body weight/dose, about 0.001 mgCa²⁺/kg body weight/dose, about 0.005 mg Ca²⁺/kg body weight/dose, about0.01 mg Ca²⁺/kg body weight/dose, about 0.02 mg Ca²⁺/kg bodyweight/dose, or about 0.5 mg Ca²⁺/kg body weight/dose.

In other embodiments the amount of calcium delivered to the upperrespiratory tract (e.g., nasal cavity) is of about 0.001 mg Ca²⁺/kg bodyweight/dose to about 2 mg Ca²⁺/kg body weight/dose, about 0.002 mgCa²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg body weight/dose, about0.005 mg Ca²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg bodyweight/dose, about 0.01 mg Ca²⁺/kg body weight/dose to about 2 mgCa²⁺/kg body weight/dose, about 0.01 mg Ca²⁺/kg body weight/dose toabout 60 mg Ca²⁺/kg body weight/dose, about 0.01 mg Ca²⁺/kg bodyweight/dose to about 50 mg Ca²⁺/kg body weight/dose, about 0.01 mgCa²⁺/kg body weight/dose to about 40 mg Ca²⁺/kg body weight/dose, about0.01 mg Ca²⁺/kg body weight/dose to about 30 mg Ca²⁺/kg bodyweight/dose, about 0.01 mg Ca²⁺/kg body weight/dose to about 20 mgCa²⁺/kg body weight/dose, about 0.01 mg Ca²⁺/kg body weight/dose toabout 10 mg Ca²⁺/kg body weight/dose, about 0.01 mg Ca²⁺/kg bodyweight/dose to about 5 mg Ca²⁺/kg body weight/dose, about 0.01 mgCa²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg body weight/dose, about0.02 mg Ca²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg body weight/dose,about 0.03 mg Ca²⁺/kg body weight/dose to about 2 mg Ca²⁺/kg bodyweight/dose, about 0.04 mg Ca²⁺/kg body weight/dose to about 2 mgCa²⁺/kg body weight/dose, about 0.05 mg Ca²⁺/kg body weight/dose toabout 2 mg Ca²⁺/kg body weight/dose, about 0.1 mg Ca²⁺/kg bodyweight/dose to about 2 mg Ca²⁺/kg body weight/dose, about 0.1 mg Ca²⁺/kgbody weight/dose to about 1 mg Ca²⁺/kg body weight/dose, about 0.1 mgCa²⁺/kg body weight/dose to about 0.5 mg Ca²⁺/kg body weight/dose, about0.2 mg Ca²⁺/kg body weight/dose to about 0.5 mg Ca²⁺/kg bodyweight/dose, about 0.18 mg Ca²⁺/kg body weight/dose, about 0.001 mgCa²⁺/kg body

In addition, when the respirable dry particles and dry powders include asodium salt, the respirable dry particles and dry powders can beadministered in an amount sufficient to deliver a dose of about 0.001 mgNa⁺/kg body weight/dose to about 10 mg Na⁺/kg body weight/dose, or about0.01 mg Na⁺/kg body weight/dose to about 10 mg Na⁺/kg body weight/dose,or about 0.1 mg Na⁺/kg body weight/dose to about 10 mg Na⁺/kg bodyweight/dose, or about 1.0 mg Na⁺/kg body weight/dose to about 10 mgNa⁺/kg body weight/dose, or about 0.001 mg Na⁺/kg body weight/dose toabout 1 mg Na⁺/kg body weight/dose, or about 0.01 mg Na⁺/kg bodyweight/dose to about 1 mg Na⁺/kg body weight/dose, or about 0.1 mgNa⁺/kg body weight/dose to about 1 mg Na⁺/kg body weight/dose, about 0.2to about 0.8 mg Na⁺/kg body weight/dose, about 0.3 to about 0.7 mgNa⁺/kg body weight/dose, or about 0.4 to about 0.6 mg Na⁺/kg bodyweight/dose.

In some embodiments the amount of sodium delivered to the respiratorytract (e.g., lungs, respiratory airway) is about 0.001 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.01 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.1 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 1 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.001 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.01 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.1 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.2 toabout 0.8 mg/kg body weight/dose, or about 0.3 to about 0.7 mg/kg bodyweight/dose, or about 0.4 to about 0.6 mg/kg body weight/dose.

In other embodiments the amount of sodium delivered to the upperrespiratory tract (e.g., nasal cavity) is about 0.001 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.01 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.1 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 1 mg/kg bodyweight/dose to about 10 mg/kg body weight/dose, or about 0.001 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.01 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.1 mg/kgbody weight/dose to about 1 mg/kg body weight/dose, or about 0.2 toabout 0.8 mg/kg body weight/dose, or about 0.3 to about 0.7 mg/kg bodyweight/dose, or about 0.4 to about 0.6 mg/kg body weight/dose.

Suitable intervals between doses that provide the desired therapeuticeffect can be determined based on the severity of the condition (e.g.,infection), overall well being of the subject and the subject'stolerance to respirable dry particles and dry powders and otherconsiderations. Based on these and other considerations, a clinician candetermine appropriate intervals between doses. Generally, respirable dryparticles and dry powders are administered once, twice or three times aday, as needed.

If desired or indicated, the respirable dry particles and dry powdersdescribed herein can be administered with one or more other therapeuticagents. The other therapeutic agents can be administered by any suitableroute, such as orally, parenterally (e.g., intravenous, intraarterial,intramuscular, or subcutaneous injection), topically, by inhalation(e.g., intrabronchial, intranasal or oral inhalation, intranasal drops),rectally, vaginally, and the like. The respirable dry particles and drypowders can be administered before, substantially concurrently with, orsubsequent to administration of the other therapeutic agent. Preferably,the respirable dry particles and dry powders and the other therapeuticagent are administered so as to provide substantial overlap of theirpharmacologic activities.

Another advantage provided by the respirable dry powders and respirabledry particles described herein, is that dosing efficiency can beincreased as a result of hygroscopic growth of particles inside thelungs, due to particle moisture growth. The propensity of the partiallyamorphous, high salt compositions of the invention to take up water atelevated humidities can also be advantageous with respect to theirdeposition profiles in vivo. Due to their rapid water uptake at highhumidities, these powder formulations can undergo hygroscopic growth dothe absorbance of water from the humid air in the respiratory tract asthey transit into the lungs. This can result in an increase in theireffective aerodynamic diameters during transit into the lungs, whichwill further facilitate their deposition in the airways.

EXEMPLIFICATION

Materials used in the following Examples and their sources are listedbelow. Calcium chloride dihydrate, calcium lactate pentahydrate, sodiumchloride, L-leucine, maltodextrin, mannitol, lactose and trehalose wereobtained from Sigma-Aldrich Co. (St. Louis, Mo.) or Spectrum Chemicals(Gardena, Calif.); sodium sulfate from EMD Chemicals (Gibbstown, N.J.),Sigma-Aldrich Co. (St. Louis, Mo.) or Spectrum Chemicals (Gardena,Calif.); and sodium citrate dihydrate from J.T. Baker (Phillipsburg,N.J.), Mallinckrodt Baker (Phillipsburg, N.J.) or Spectrum Chemicals(Gardena, Calif.). Ultrapure water was from a water purification system(Millipore Corp., Billerica, Mass.).

Methods:

Geometric or Volume Diameter.

Volume median diameter (×50 or Dv50), which may also be referred to asvolume median geometric diameter (VMGD), was determined using a laserdiffraction technique. The equipment consisted of a HELOS diffractometerand a RODOS dry powder disperser (Sympatec, Inc., Princeton, N.J.). TheRODOS disperser applies a shear force to a sample of particles,controlled by the regulator pressure (typically set at 1.0 bar) of theincoming compressed dry air. The pressure settings may be varied to varythe amount of energy used to disperse the powder. For example, theregulator pressure may be varied from 0.2 bar to 4.0 bar; and theorifice ring pressure may be varied from 5.00 mbar to 115.00 mbar.Powder sample is dispensed from a microspatula into the RODOS funnel.The dispersed particles travel through a laser beam where the resultingdiffracted light pattern produced is collected, typically using an R1lens, by a series of detectors. The ensemble diffraction pattern is thentranslated into a volume-based particle size distribution using theFraunhofer diffraction model, on the basis that smaller particlesdiffract light at larger angles. Using this method geometric standarddeviation (GSD) for the volume mean geometric diameter was alsodetermined.

Volume median diameter can also be measured using a method where thepowder is emitted from a dry powder inhaler device. The equipmentconsisted of a Spraytec laser diffraction particle size system (Malvern,Worcestershire, UK), “Spraytec”. Powder formulations were filled intosize 3 HPMC capsules (Capsugel V-Caps) by hand with the fill weightmeasured gravimetrically using an analytical balance (Mettler TolerdoXS205). A capsule based passive dry powder inhalers (RS-01 Model 7, Highresistance Plastiape S.p.A.) was used which had specific resistance of0.036 kPa^(1/2) LPM⁻¹. Flow rate and inhaled volume were set using atimer controlled solenoid valve with flow control valve (TPK2000, CopleyScientific). Capsules were placed in the dry powder inhaler, puncturedand the inhaler sealed to the inlet of the laser diffraction particlesizer. The steady air flow rate through the system was initiated usingthe TPK2000 and the particle size distribution was measured via theSpraytec at 1 kHz for at least 2 seconds and up to the total inhalationduration. Particle size distribution parameters calculated included thevolume median diameter (Dv50) and the geometric standard deviation (GSD)and the fine particle fraction (FPF) of particles less than 5micrometers in diameter. At the completion of the inhalation duration,the dry powder inhaler was opened, the capsule removed and re-weighed tocalculate the mass of powder that had been emitted from the capsuleduring the inhalation duration (capsule emitted powder mass or CEPM).

The previous description of the use of the Spraytec was for what isdescribed as its “closed bench configuration”. Alternatively, theSpraytec can be used in its “open bench configuration”. In the openbench configuration, capsules were placed in the dry powder inhaler,punctured and the inhaler sealed inside a cylinder. The cylinder wasconnected to a positive pressure air source with steady air flow throughthe system again measured with a mass flow meter and its durationcontrolled with a timer controlled solenoid valve. The exit of the drypowder inhaler was exposed to room pressure and the resulting aerosoljet passed through the laser of the diffraction particle sizer(Spraytec) in its open bench configuration before being captured by avacuum extractor. The steady air flow rate through the system wasinitiated using the solenoid valve and the particle size distributionwas measured via the Spraytec at 1 kHz for the duration of the singleinhalation maneuver with a minimum of 2 seconds, as in the closed benchconfiguration. When data are reported in the examples as being measuredby the Spraytec, they are from the closed bench configuration unlessotherwise noted.

Emitted Geometric or Volume Diameter.

The volume median diameter (Dv50) of the powder after it emitted from adry powder inhaler, which may also be referred to as volume mediangeometric diameter (VMGD), was determined using a laser diffractiontechnique via the Spraytec diffractometer (Malvern, Inc.,Worcestershire, UK). Powder was filled into size 3 capsules (V-Caps,Capsugel) and placed in a capsule based dry powder inhaler (RS01 Model 7High resistance, Plastiape, Italy), or DPI, which was connected with anairtight seal to the inhaler adapter of the Spraytec. A steady airflowrate was drawn through the DPI typically at 60 L/min for a set duration,typically of 2 seconds controlled by a timer controlled solenoid(TPK2000, Copley, Scientific, UK). Alternatively, the airflow rate drawnthrough the DPI was sometimes run at 15 L/min, 20 L/min, or 30 L/min.The outlet aerosol then passed perpendicularly through the laser beam asan internal flow. The resulting geometric particle size distribution ofthe aerosol was calculated from the software based on the measuredscatter pattern on the photodetectors with samples typically taken at1000 Hz for the duration of the inhalation. The Dv50, GSD, FPF<5.0 μmmeasured were then averaged over the duration of the inhalation.

Fine Particle Fraction.

The aerodynamic properties of the powders dispersed from an inhalerdevice were assessed with a Mk-II 1 ACFM Andersen Cascade Impactor(Copley Scientific Limited, Nottingham, UK). The instrument was run incontrolled environmental conditions of 22±2° C. and relative humidity(RH) between 30±5%. The instrument consists of eight stages thatseparate aerosol particles based on inertial impaction. At each stage,the aerosol stream passes through a set of nozzles and impinges on acorresponding impaction plate. Particles having small enough inertiawill continue with the aerosol stream to the next stage, while theremaining particles will impact upon the plate. At each successivestage, the aerosol passes through nozzles at a higher velocity andaerodynamically smaller particles are collected on the plate. After theaerosol passes through the final stage, a filter collects the smallestparticles that remain, called the “final collection filter”. Gravimetricand/or chemical analyses can then be performed to determine the particlesize distribution. A short stack cascade impactor, also referred to as acollapsed cascade impactor, is also utilized to allow for reduced labortime to evaluate two aerodynamic particle size cut-points. With thiscollapsed cascade impactor, stages are eliminated except those requiredto establish fine and coarse particle fractions.

The impaction techniques utilized allowed for the collection of two oreight separate powder fractions. The capsules (HPMC, Size 3; ShionogiQualicaps, Madrid, Spain or Capsugel Vcaps, Peapack, N.J.) wereapproximately half-filled with powder and placed in a hand-held,breath-activated dry powder inhaler (DPI) device, the high resistanceRS-01 DPI (Plastiape, Osnago, Italy). The capsule was punctured and thepowder was drawn through the cascade impactor operated at a flow rate of60.0 L/min for 2.0 s. At this flowrate, the calibrated cut-off diametersfor the eight stages are 8.6, 6.5, 4.4, 3.3, 2.0, 1.1, 0.5 and 0.3microns and for the two stages used with the short stack cascadeimpactor, the cut-off diameters are 5.6 microns and 3.4 microns. Thefractions were collected by placing filters in the apparatus anddetermining the amount of powder that impinged on them by gravimetricmeasurements or chemical measurements on an HPLC, as labeled in thetables. The fine particle fraction of the total dose of powder (FPF_TD)less than or equal to an effective cut-off aerodynamic diameter wascalculated by dividing the powder mass recovered from the desired stagesof the impactor by the total particle mass in the capsule. Results arereported as the fine particle fraction of less than 5.6 microns (FPF<5.6 microns) and the fine particle fraction of less than 3.4 microns(FPF <3.4 microns). The fine particle fraction can alternatively becalculated relative to the recovered or emitted dose of powder bydividing the powder mass recovered from the desired stages of theimpactor by the total powder mass recovered.

Aerodynamic Diameter.

Mass median aerodynamic diameter (MMAD) was determined using theinformation obtained by the Andersen Cascade Impactor. The cumulativemass under the stage cut-off diameter is calculated for each stage andnormalized by the recovered dose of powder. The MMAD of the powder isthen calculated by linear interpolation of the stage cut-off diametersthat bracket the 50th percentile.

Fine Particle Dose.

The fine particle dose was determined using the information obtained bythe ACI. The cumulative mass deposited on the final collection filter,and stages 6, 5, 4, 3, and 2 for a single dose of powder actuated intothe ACI is equal to the fine particle dose less than 4.4 microns(FPD<4.4 μm).

Capsule Emitted Powder Mass.

A measure of the emission properties of the powders was determined byusing the information obtained from the Andersen Cascade Impactor tests.The filled capsule weight was recorded at the beginning of the run andthe final capsule weight was recorded after the completion of the run.The difference in weight represented the amount of powder emitted fromthe capsule (CEPM or capsule emitted powder mass). The emitted dose wascalculated by dividing the amount of powder emitted from the capsule bythe total initial particle mass in the capsule. While the standard CEPMwas measured at 60 L/min, it was also measured at 15 L/min, 20 L/min, or30 L/min.

Tap Density.

Two methods were utilized to measure tap density. (1) A modified methodrequiring smaller powder quantities was initially used, following USP<616> with the substitution of a 1.5 cc microcentrifuge tube (EppendorfAG, Hamburg, Germany) or a 0.3 cc section of a disposable serologicalpolystyrene micropipette (Grenier Bio-One, Monroe, N.C.) withpolyethylene caps (Kimble Chase, Vineland, N.J.) to cap both ends andhold the powder. (2) USP <616> was used, utilizing a 100 cc graduatedcylinder. Instruments for measuring tap density, known to those skilledin the art, include but are not limited to the Dual PlatformMicroprocessor Controlled Tap Density Tester (Vankel, Cary, N.C.) or aGeoPyc instrument (Micrometrics Instrument Corp., Norcross, Ga.). Tapdensity is a standard, approximated measure of the envelope massdensity. The envelope mass density of an isotropic particle is definedas the mass of the particle divided by the minimum spherical envelopevolume within which it can be enclosed.

Bulk Density.

Bulk density was estimated prior to tap density measurement procedure bydividing the weight of the powder by the volume of the powder, asestimated using the volumetric measuring device.

Hausner Ratio.

This is a dimensionless number, which was calculated by dividing the tapdensity by the bulk density. It is a number that is correlated to theflowability of a powder.

Scanning Electron Microscopy (SEM).

SEM was performed using a FE Quanta 200 scanning electron microscope(Hillsboro, Oreg.) equipped with an Everhart Thornley (ET) detector.Images were collected and analysed using xTm (v. 2.01) and XT Docu (v.3.2) software, respectively. The magnification was verified using a NISTtraceable standard. Each sample was prepared for analysis by placing asmall amount on a carbon adhesive tab supported on an aluminum mount.Each sample was then sputter coated with Au/Pd using a Cressington 108auto Sputter Coater at approximately 20 mA and 0.13 mbar (Ar) for 75seconds. The data acquisition parameters are displayed in theinformation bar at the bottom of each image. The magnification reportedon each image was calculated upon the initial data acquisition. Thescale bar reported in the lower portion of each image is accurate uponresizing and should be used when making size determinations.

Liquid Feedstock Preparation for Spray Drying.

Spray drying homogenous particles requires that the ingredients ofinterest be solubilized in solution or suspended in a uniform and stablesuspension. Certain calcium salts, such as calcium chloride, calciumacetate and calcium lactate, are sufficiently water-soluble to preparesuitable spray drying solutions. However, other calcium salts, such ascalcium sulfate, calcium citrate and calcium carbonate, have a lowsolubility in water. The solubility in water of exemplary calcium saltsare listed in Table 1. As a result of these low solubilities,formulation feedstock development work was necessary to preparesolutions or suspensions that could be spray dried. These solutions orsuspensions included combinations of salts in an appropriate solvent,typically water but also ethanol and water mixtures or other solvents asdescribed earlier in the specification.

TABLE 1 Calcium Salts’ Solubility in Water Calcium Salt Solubility inWater (at 20-30° C., 1 bar) Salt Water solubility (g/L) Calcium chloride 1368^(1.2) Calcium acetate 347¹ Calcium lactate 105¹ Calcium gluconate33.23³   Calcium sulate 2.98¹ Calcium citrate 0.96¹ Calcium phosphatedibasic  0.2¹ Calcium carbonate Pract. Insol.² Calcium stearate Pract.Insol.² Calcium alginate Not applicable Sodium Carbonate 505¹ SodiumChloride 360¹ Sodium Citrate 910¹ Sodium Sulfate 194¹ ¹Perry, Robert H.,Don W. Green, and James O. Maloney. Perry's Chemical Engineers’Handbook, 7th ed. New York: McGraw-Hill, 1997, Print. ²Solubility at 60°C. ³O'Neil, Maryadele J. The Merck Index: an Encyclopedia of Chemicals,Drugs, and Biologicals, 14th ed. Whitehouse Station, N.J.: Merck, 2006.Print.

As mentioned previously, calcium chloride has high water solubility.Sodium salts, such as sodium sulfate, sodium citrate and sodiumcarbonate, are also very soluble in water. As will be discussed furtherin the following examples, calcium chloride and sodium salts (the“starting materials”) are combined in solution or suspension to obtainstable calcium salts in final dry powder form. When combining thecalcium chloride and sodium salt in solution, the calcium and the anioncontributed from the sodium salt may react in a precipitation reactionto produce the desired calcium salt (i.e., CaCl₂+2NaXX→CaXX+2NaCl). Inthis case, the maximum solids concentration that maintained a clearsolution or a stable suspension were used for spray drying. Certaincalcium salts were soluble enough to be dissolved in water and thenspray dried alone. The same concept may be applied to, for example,magnesium salts by using magnesium chloride, potassium salts usingpotassium chloride, and sodium salts.

The starting materials may be provided in molar amounts where the fullprecipitation reaction may proceed to completion, termed ‘reaction tocompletion.’ The weight percent of calcium ion in exemplary calciumsalts are further listed in Table 2.

TABLE 2 Weight Percent of Ca²⁺ in Salt Molecules Weight % of Calcium ionin Salt Molecule Weight % of Ca²⁺ in Salt Formula MW molecule Calciumcarbonate CaCO₃ 100.09 40.0 Calcium chloride CaCl₂ 110.98 36.0 Calciumphosphate dibasic CaHPO₄ 136.06 29.4 Calcium sulfate CaSO₄ 136.14 29.4Calcium acetate Ca(C₂H₃O₂)₂ 158.17 25.3 Calcium citrate Ca₃(C₆H₅O₇)₂498.46 24.1 Calcium lactate Ca(C₃H₅O₃)₂ 218.218 18.3 Calcium sorbateCaC₁₂H₁₄O₄ 262.33 15.2 Calcium gluconate CaC₁₂H₂₂O₁₄ 430.373 9.3 Calciumstearate CaC₃₆H₇₀O₄ 607.02 6.6 Calcium alginate [Ca(C₆H₇O₆)₂]_(n) NA NA

Alternatively, excess calcium chloride may be added for an incompletereaction, or ‘reaction not to completion,’ where a given amount ofcalcium chloride is present in the final powder form. While calciumchloride is hygroscopic, its high water solubility may be beneficial tohave in small amounts in the final product to increase the solubility ofthe final product, to be able to tailor the dissolution profile, and toincrease the relative calcium ion ratio to sodium or other cationspresent in the formulation. For ease of formulation development, therequired molar ratios of calcium chloride and sodium salt were convertedto mass ratios of calcium chloride and sodium salt. An example is forcalcium citrate (i.e., calcium chloride+sodium citrate), where theprecipitation reaction proceeds forward as follows:3CaCl₂+2Na₃C₆H₅O₇→Ca₃(C₆H₅O₇)₂+6NaCl

This reaction results in a 1:2 molar ratio of Ca:Na ions. For thereaction to proceed to completion, 3 moles of calcium chloride and 2moles of sodium citrate are required. To convert to mass in grams and aweight ratio, the moles of salts are multiplied by the molecular weightof the salts in grams per mole:For calcium chloride: 3 mol CaCl₂×111 g/mol=333 g CaCl₂For sodium citrate: 2 mol Na₃C₆H₅O₇×258 g/mol=516 g Na₃C₆H₅O₇

Therefore, a 1:1.55 or 39:61 weight ratio of CaCl₂:Na₃C₆H₅O₇ is requiredfor a complete reaction. These ratios were solubilized and spray driedto produce ‘pure salt’ formulations. In addition, dry powders wereproduced with an additional excipient, such as leucine or lactose. Theratio of calcium to sodium salt remained the same so as to produce a‘reaction to completion.’ For example, for a formulation of 50% (w/w)leucine, the remainder is composed of salts, such as calcium citrate(i.e., CaCl₂:Na₃C₆H₅O₇) where the 39:61, CaCl₂:Na₃C₆H₅O₇ weight ratio ismaintained. Thus, for that reaction: 50% (w/w) leucine, 19.5% (w/w)CaCl₂ and 30.5% (w/w) Na₃C₆H₅O₇ will be added. For a spray dryingprocess, the salts and other excipients will be dissolved or suspendedin a solvent (i.e., water). The solids concentration (w/v) can be chosendepending on the solubility of the different components. For the citrateformulation, a concentration of 5 mg/mL was appropriate, given thelimited solubility of calcium citrate: 0.95 mg/mL. Therefore, 5 g ofsolids (i.e., 2.5 g leucine, 0.975 g calcium chloride and 1.525 g ofsodium citrate) were dissolved in 1 L of ultrapure water.

In addition, when preparing spray drying solutions, the water weight ofthe hydrated starting material must be accounted for. The ratios usedfor formulations were based on the molecular weight of the anhydroussalts. For certain salts, hydrated forms are more readily available thanthe anhydrous form. This required an adjustment in the ratios originallycalculated, using a multiplier to correlate the molecular weight of theanhydrous salt with the molecular weight of the hydrate. An example ofthis calculation is included below.

For the example above, calcium chloride anhydrous molecular weight is110.98 g/mol and the dihydrate molecular weight is 147.01 g/mol. Sodiumcitrate anhydrous molecular weight is 258.07 g/mol and the dihydratemolecular weight is 294.10 g/mol.

The multiplier is analogous to the ratio of the dihydrate to anhydrousmolecular weight, e.g., 1.32 for calcium chloride and 1.14 for sodiumcitrate. Therefore, adjusting for the dihydrate forms results in: 2.5 gleucine, 1.287 g (i.e., 0.975 g×1.32) calcium chloride dihydrate and1.738 g (i.e., 1.525 g×1.14) of sodium citrate dihydrate were dissolvedand spray dried.

Spray Drying Using Niro Spray Dryer.

Dry powders were produced by spray drying utilizing a Niro Mobile Minorspray dryer (GEA Process Engineering Inc., Columbia, Md.) with powdercollection from a cyclone, a product filter or both. Atomization of theliquid feed was performed using a co-current two-fluid nozzle eitherfrom Niro (GEA Process Engineering Inc., Columbia, Md.) or a SprayingSystems (Carol Stream, Ill.) two-fluid nozzle with gas cap 67147 andfluid cap 2850SS, although other two-fluid nozzle setups are alsopossible. For example, the two-fluid nozzle can be in an internal mixingsetup or an external mixing setup. Additional atomization techniquesinclude rotary atomization or a pressure nozzle. The liquid feed was fedusing gear pumps (Cole-Parmer Instrument Company, Vernon Hills, Ill.)directly into the two-fluid nozzle or into a static mixer (Charles Ross& Son Company, Hauppauge, N.Y.) immediately before introduction into thetwo-fluid nozzle. An additional liquid feed technique includes feedingfrom a pressurized vessel. Nitrogen or air may be used as the dryinggas, provided that moisture in the air is at least partially removedbefore its use. Pressurized nitrogen or air can be used as theatomization gas feed to the two-fluid nozzle. The process gas inlettemperature can range from 100° C. to 300° C. and outlet temperaturefrom 50° C. to 120° C. with a liquid feedstock rate of 20 mL/min to 100mL/min. The gas supplying the two-fluid atomizer can vary depending onnozzle selection and for the Niro co-current two-fluid nozzle can rangefrom 8 kg/hr to 15 kg/hr and be set a pressures ranging from 0.5 bar to2.0 bar or for the Spraying Systems two-fluid nozzle with gas cap 67147and fluid cap 2850SS can range from 40 to 100 g/min. For example, theNiro two fluid nozzle discussed above can range from 5 kg/hr to 50kg/hr. The atomizing gas rate can be set to achieve a certain gas toliquid mass ratio, which directly affects the droplet size created. Thepressure inside the drying drum can range from +3 “WC to −6 “WC. Spraydried powders can be collected in a container at the outlet of thecyclone, onto a cartridge or baghouse filter, or from both a cyclone anda cartridge or baghouse filter.

Spray Drying Using Büchi Spray Dryer.

Dry powders were prepared by spray drying on a Büchi B-290 Mini SprayDryer (BÜCHI Labortechnik AG, Flawil, Switzerland) with powdercollection from either a standard or High Performance cyclone. Thesystem used the Büchi B-296 dehumidifier to ensure stable temperatureand humidity of the air used to spray dry. Furthermore, when therelative humidity in the room exceeded 30% RH, an external LGdehumidifier (model 49007903, LG Electronics, Englewood Cliffs, N.J.)was run constantly. Atomization of the liquid feed utilized a Büchitwo-fluid nozzle with a 1.5 mm diameter. Inlet temperature of theprocess gas can range from 100° C. to 220° C. and outlet temperaturefrom 80° C. to 120° C. with a liquid feedstock flowrate of 3 mL/min to10 mL/min. The two-fluid atomizing gas ranges from 25 mm to 45 mm (300LPH to 530 LPH) and the aspirator rate from 70% to 100% (28 m³/hr to 38m³/hr).

Table 3 provides feedstock formulations used in preparation of some drypowders described herein.

TABLE 3 Feedstock Formulations Ca:Na molar Formulation FeedstockComposition (w/w) ratio I 10.0% leucine, 35.1% calcium chloride, 54.9%sodium 1:2 citrate [12.7% Ca²⁺ (w/w); 14.7% Na⁺ (w/w)] II 10.0% leucine,39.6% calcium chloride, 50.4% sodium 1:2 sulfate [14.3% Ca²⁺ (w/w); 8.2%Na⁺ (w/w)] III 10.0% leucine, 58.6% calcium lactate, 31.4% sodium 1:2chloride [10.8% Ca²⁺ (w/w); 12.4% Na⁺ (w/w)] IV 10.0% maltodextrin,58.6% calcium lactate, 31.4% 1:2 sodium chloride [10.8% Ca²⁺ (w/w);12.4% Na⁺ (w/w)] V 10.0% mannitol, 58.6% calcium lactate, 31.4% sodium1:2 chloride [10.8% Ca²⁺ (w/w); 12.4% Na⁺ (w/w)] VI 39.4% leucine, 58.6%calcium lactate, 2.0% sodium 8:1 chloride [10.8% Ca²⁺ (w/w); 0.8% Na⁺(w/w)] VII 37.5% leucine, 58.6% calcium lactate, 3.9% sodium 4:1chloride [10.8% Ca²⁺ (w/w); 1.5% Na⁺ (w/w)] VIII 20% leucine, 75.0%calcium lactate, 5.0% sodium 4:1 chloride [13.8% Ca²⁺ (w/w); 2.0% Na⁺(w/w)] IX 33.6% leucine, 58.6% calcium lactate, 7,8% sodium chloride[10.8% Ca²⁺ (w/w); 3.1% Na⁺ (w/w)]Table 4 provides expected final dry powder compositions. Thesecompositions are based on the expectation that the ion exchange reactiondescribed above goes to completion for Formulations I and III. Withoutwishing to be bound by any particular theory, the evaporation of thedroplet that occurs during spray drying is expected to drive the leastsoluble salt to precipitate first, which is the calcium citrate andcalcium sulfate in Formulations I and II, respectively.

TABLE 4 Dry Powder Products of Spray Drying Formulation Composition(w/w) I 10.0% leucine, 52.8% calcium citrate, 37.2% sodium chloride II10.0% leucine, 48.4% calcium sulfate, 41.6% sodium chloride III 10.0%leucine, 58.6% calcium lactate, 31.4% sodium chloride IV 10.0%maltodextrin, 58.6% calcium lactate, 31.4% sodium chloride V 10.0%mannitol, 58.6% calcium lactate, 31.4% sodium chloride VI 39.4% leucine,58.6% calcium lactate, 2.0% sodium chloride VII 37.5% leucine, 58.6%calcium lactate, 3.9% sodium chloride VIII 20% leucine, 75.0% calciumlactate, 5.0% sodium chlorideDescription of Placebo:

Placebo formulations comprising 100 weight percent leucine or 98 weightpercent leucine with 2 weight percent sodium chloride were produced byspray drying. An aqueous phase was prepared for a batch process bydissolving leucine in ultrapure water with constant agitation until thematerials were completely dissolved in the water at room temperature.For a static mixing process, the ultrapure water was divided in half andhalf of the total required leucine was dissolved in each volume ofwater. The solutions were then spray dried using a Niro or a Büchi spraydryer. For the Placebo formulation, two batches (A and B) of feedstockwere prepared and spray dried. The total solids concentration for BatchA was 15 g/L and for Batch B was 5 g/L. The process conditions used forspray drying Batch A (Placebo-A) on the Niro Mobile Minor spray dryerwere similar to the conditions used to spray dry Formulation 1-A inExample 1. The process conditions used for spray drying Batch B(Placebo-B) were similar to the conditions used to spray dry FormulationI-C in Example 1, with the exception that the outlet temperature wasabout 82° C. for Formulation Placebo-B. Additional information relatingto process conditions and properties of the Formulation Placebo-A andPlacebo-B powders and/or particles prepared in this example are providedin the Tables or graphs shown in FIGS. 1A-1F and 2-4.

Example 1

This example describes the preparation of dry powders using feedstock ofFormulation I: 10.0 weight percent leucine, 35.1 weight percent calciumchloride and 54.9 weight percent sodium citrate.

An aqueous phase was prepared for a hatch process by dissolving leucinein ultrapure water, then sodium citrate dihydrate, and finally calciumchloride dihydrate. The solution or suspension was kept agitatedthroughout the process until the materials were completely dissolved inthe water at room temperature. For a static mixing process, the sodiumsalt and calcium salt were kept in separate solutions. The ultrapurewater was divided in half and half of the total required leucine wasdissolved in each volume of water. The sodium citrate dihydrate wasdissolved in one aqueous phase and the calcium chloride dihydratedissolved in the second aqueous phase. The solutions or suspensions werekept agitated throughout the process until the materials were completelydissolved in the water at room temperature. The solutions or suspensionswere then spray dried using a Niro or a Büchi spray dryer. For eachformulation, three batches (A, B & C) of feedstock were prepared andspray dried. Details on the liquid feedstock preparations for each ofthe three hatches are shown in Table 5, where the total solidsconcentration is reported as the total of the dissolved anhydrousmaterial weights. Batch A and D particles were prepared using batch Aand D feedstock, respectively, on a Niro spray dryer. Batch B and Cparticles were prepared using the corresponding feedstocks on a Büchispray dryer.

TABLE 5 Summary of liquid feedstock preparations of four batches ofparticles for Formulation I. Formulation: I-A I-B I-C I-D Liquidfeedstock mixing Static Batch Batch Static mixed mixed mixed mixed Totalsolids concentration 10 g/L 5 g/L 5 g/L 15 g/L Total solids 380 g 6.25 g10.50 g 570 g Total volume water 38.0 L 1.25 L 2.1 L 38 L Amount leucinein 1 L 1.00 g 0.50 g 1.05 g 1.5 g Amount sodium citrate 6.26 g 3.13 g3.13 g 9.39 g dihydrate in 1 L Amount calcium chloride 4.65 g 2.32 g2.32 g 6.98 g dihydrate in 1 L

Batch A (I-A) dry powders were produced by spray drying on the NiroMobile Minor spray dryer (GEA Process Engineering Inc., Columbia, Md.)with powder collection from a product cartridge filter. Atomization ofthe liquid feed used a co-current two-fluid nozzle from Niro (GEAProcess Engineering Inc., Columbia, Md.) with 1.0 mm insert. The liquidfeed was fed using gear pumps (Cole-Parmer Instrument Company, VernonHills, Ill.) into a static mixer (Charles Ross & Son Company, Hauppauge,N.Y.) immediately before introduction into the two-fluid nozzle.Nitrogen was used as the drying gas. The process gas inlet temperaturewas set to 282° C., with the outlet temperature reading about 98° C. Thegas supplying the two-fluid atomizer was set at a flowrate of 14.5 kg/hrand a pressure of 2 psi, the process gas flowrate was set at 85 kg/hrand a pressure of 25 psi, and the pressure inside the drying drum was at−2 “WC. The liquid feed stock total flowrate was 70 mL/min, with eachstream being fed at 35 mL/min. Spray dried powders were collected from aproduct collection cartridge filter.

Batch B (I-B) and Batch C (I-C) dry powders were prepared by spraydrying on a Büchi B-290 Mini Spray Dryer (BÜCHI Labortechnik AG, Flawil,Switzerland) with a Büchi two-fluid nozzle with a 1.5 mm diameter andpowder collection from a High Performance cyclone. The system used theBüchi B-296 dehumidifier to ensure stable temperature and humidity ofthe air used to spray dry. Inlet temperature of the process gas was setat 220° C. with a liquid feedstock flowrate of 6.7 mL/min forFormulation I-B and 7 mL/min for Formulation I-C. The outlet temperaturewas about 108° C. for Formulation I-B and about 95° C. for FormulationI-C. The two-fluid atomizing gas was at 40 mm and the aspirator rate at90%.

Batch D (I-D) dry powders were produced by spray drying on the NiroMobile Minor spray dryer (GEA Process Engineering Inc., Columbia, Md.)with powder collection from a product filter membrane. Atomization ofthe liquid feed used a two-fluid nozzle from Spraying Systems (CarolStream, II,) with gas cap 67147 and fluid cap 2850SS. The liquid feedwas fed using gear pumps (Cole-Parmer Instrument Company, Vernon Hills,Ill.) into a static mixer (Charles Ross & Son Company, Hauppauge, N.Y.)immediately before introduction into the two-fluid nozzle. Nitrogen wasused as the drying gas. The process gas inlet temperature was set toapproximately 265° C., with the outlet temperature reading about 99° C.The gas supplying the two-fluid atomizer was set at a flowrate of 80g/min, the process gas flowrate was set at 80 kg/hr and the pressureinside the drying drum was at −2 “WC. The liquid feed stock totalflowrate was 66 mL/min, with each stream being fed at 33 mL/min. Spraydried powders were collected from a product collection filter membrane.

Some of the physical properties of the particles obtained in fourseparate hatches (Formulation I-A, I-B, I-C and I-D) are summarized inTable 6. In addition to the data provided in Table 5, further datarelated to the dry powders prepared from feedstock formulation I-A issummarized as follows. The fine particle fraction (FPF) as measured by afull 8-stage Andersen Cascade Impactor with gravimetric analysis was onaverage 56.2% for FPF less than 5.6 microns and 41.7% for FPF less than3.4 microns. The aerodynamic diameter was also measured with afull-stage ACI with gravimetric analysis. The average value for the massmedian aerodynamic diameter (MMAD) was 2.72 microns. The volume size wasdetermined by laser diffraction on the HELOS/RODOS sizing equipment andthe average value for the volume median diameter (×50) at a pressure of1 bar was 2.57 microns. In addition, the powder displayed relativelyflowrate independent behavior as can be seen from the ratio of ×50measured at 0.5 bar to ×50 measured at 4.0 bar, which was 1.19. Thevalue for 1/4 bar for these particles was 1.17.

Additional properties of the dry powders prepared from feedstockFormulation I-D are summarized as follows. The fine particle fraction(FPF) as measured by a full 8-stage Andersen Cascade Impactor withgravimetric analysis was on average 58.8% for FPF less than 5.6 micronsand 46.7% for FPF less than 3.4 microns. The aerodynamic diameter wasalso measured with a full-stage ACI with gravimetric analysis. Theaverage value for the mass median aerodynamic diameter (MMAD) was 2.38microns. The volume size was determined by laser diffraction on theHELOS/RODOS sizing equipment and the average value for the volume mediandiameter (×50) at a pressure of 1 bar was 2.45 microns. In addition, thepowder displayed relatively flowrate independent behavior as can be seenfrom the ratio of ×50 measured at 0.5 bar to ×50 measured at 4.0 bar,which was 1.12. The value for 1/4 bar for these particles was 1.09.

TABLE 6 Summary of ACI-2 data for the four batches of particles forFormulation I. Formulation: I-A I-B I-C I-D FPF less than 5.6 μm on 61.649.2 64.8 672 ACI-2 (%) FPF less than 3.4 μm on 45.7 33.3 52.1 54.8ACI-2 (%)

Additional information relating to properties of the Formulation I-Apowder and/or particles prepared in this example are provided in theTables or graphs shown in FIGS. 1A-1F and 2-4. In FIG. 1D, GSD refers togeometric standard deviation. In FIG. 1F, Dv50 refers to volume mediangeometric diameter (VMGD) as measured by Spraytec instrument; V refersto volume. SEM was performed as described above (FIG. 5A).

Example 2

This example describes the preparation of dry powders using feedstock ofFormulation III: 10.0 weight percent leucine, 58.6 weight percentcalcium lactate and 31.4 weight percent sodium chloride.

An aqueous phase was prepared for a batch process by dissolving leucinein ultrapure water, then sodium chloride, and finally calcium lactatepentahydrate. The solution was kept agitated throughout the processuntil the materials were completely dissolved in the water at roomtemperature. For the calcium lactate formulation, four batches (A, B, Cand D) of feedstock were prepared and spray dried. Details on the liquidfeedstock preparations for each of the four batches are shown in Table7, where the total solids concentration is reported as the total of thedissolved anhydrous material weights. Batch A and D particles wereprepared using batch A and D feedstock, respectively on a Niro spraydryer. The process conditions used for spray drying Batch A (III-A) weresimilar to the conditions used to spray dry Formulation I-A in Example 1and those for Batch D (III-D) were similar to the conditions used tospray dry Formulation 1-D in Example 1. Batch B and C particles wereprepared using the corresponding feedstocks on a Büchi Mini spray dryerwith process conditions similar to those used to spray dry forFormulations I-B and I-C in Example 1, with the exception of thefollowing process conditions. The liquid feedstock flowrate was set at5.2 mL/min for Formulation III-B and 6 mL/min for Formulation III-C. Theoutlet temperature was about 91° C. to 109° C. for Formulation III-B andabout 100° C. for Formulation III-C.

TABLE 7 Summary of liquid feedstock preparations of four batches ofparticles for Formulation III. Formulation: III-A III-B III-C III-DLiquid feedstock mixing Static mixed Batch mixed Batch mixed Staticmixed Total solids concentration 10 g/L 5 g/L 5 g/L 15 g/L Total solids400 g 10.0 g 9.20 g 570 g Total volume water 40.0 L 2.00 L 1.84 L 38 LAmount leucine in 1 L 1.00 g 0.50 g 0.50 g 1.5 g Amount sodium chloridein 1 L 3.14 g 1.57 g 1.57 g 4.71 g Amount calcium lactate 8.28 g 4.13 g4.13 g 12.42 g pentahydrate in 1 L

Some of the physical properties of the particles obtained in fourseparate batches (Formulation III-A, III-B, III-C and III-D) aresummarized in Table 8. In addition to the data provided in Table 8,further data about the dry particles prepared by feedstock FormulationIII-A is summarized as follows. The fine particle fraction (FPF) asmeasured by a full 8-stage Andersen Cascade Impactor with gravimetricanalysis was on average 55.3% for FPF less than 5.6 microns and 39.7%for FPF less than 3.4 microns. The aerodynamic diameter was alsomeasured with a full-stage ACI with gravimetric analysis. The averagevalue for the mass median aerodynamic diameter (MMAD) was 2.89 microns.The volume size was determined by laser diffraction on the HELOS/RODOSsizing equipment and the average value for the volume median diameter(×50) at a pressure of 1 bar was 1.51 microns. In addition, the powderdisplayed relatively flowrate independent behavior as can be seen fromthe ratio of ×50 measured at 0.5 bar to ×50 measured at 4.0 bar, whichwas 1.12. The value for 1/4 bar for these particles was 1.08.

Additional properties of the dry powders prepared by feedstockformulation III-D are summarized as follows. The fine particle fraction(FPF) as measured by a full 8-stage Andersen Cascade Impactor withgravimetric analysis was on average 62.2% for FPF less than 5.6 micronsand 45.3% for FPF less than 3.4 microns. The aerodynamic diameter wasalso measured with a full-stage ACI with gravimetric analysis. Theaverage value for the mass median aerodynamic diameter (MMAD) was 2.72microns. The volume size was determined by laser diffraction on theHELOS/RODOS sizing equipment and the average value for the volume mediandiameter (×50) at a pressure of 1 bar was 1.47 microns. In addition, thepowder displayed relatively flowrate independent behavior as can be seenfrom the ratio of ×50 measured at 0.5 bar to ×50 measured at 4.0 bar,which was 1.08. The value for 1/4 bar for these particles was 1.03.

TABLE 8 Summary of ACI-2 data for the four batches of particles forFormulation III. Formulation: III-A III-B III-C III-D FPF less than 5.6μm on ACI-2 63.5 55.4 56.5 71.4 (%) FPF less than 3.4 μm on ACI-2 43.435.5 34.7 49.7 (%)

Additional information relating to properties of the Formulation IIIpowders and/or particles prepared in this example are provided in theTables or graphs shown in FIGS. 1A-1F and 2-4. SEM was performed asdescribed above (FIG. 5B).

Example 3

This example describes the preparation of dry powders using feedstock ofFormulation II: 10 weight percent leucine, 39.6 weight percent calciumchloride and 50.4 weight percent sodium sulfate.

An aqueous phase was prepared for a batch process by dissolving leucinein ultrapure water, then sodium sulfate, and finally calcium chloridedihydrate. The solution or suspension was kept agitated throughout theprocess until the materials were completely dissolved in the water atroom temperature. For a static mixing process, the sodium salt andcalcium salt were kept in separate solutions. The ultrapure water wasdivided in half and half of the total required leucine was dissolved ineach volume of water. The sodium sulfate was dissolved in one aqueousphase and the calcium chloride dihydrate dissolved in the second aqueousphase. The solutions or suspensions were kept agitated throughout theprocess until the materials were completely dissolved in the water atroom temperature. The solutions or suspensions were then spray driedusing a Niro or a Büchi spray dryer. For each formulation, four batches(A, B, C and D) of feedstock were prepared and spray dried. Details onthe liquid feedstock preparations for each of the four batches are shownin Table 9, where the total solids concentration is reported as thetotal of the dissolved anhydrous material weights. Batch A and Dparticles were prepared using batch A and D feedstock, respectively on aNiro spray dryer. Batch B and C particles were prepared using thecorresponding feedstocks on a Büchi spray dryer. The process conditionsused for spray drying Batch A (II-A) were similar to the conditions usedto spray dry Formulation I-A in Example 1 and the process conditionsused for spray drying Batch D (II-D) were similar to the conditions usedto spray dry Formulation I-D in Example 1. Batch B and C particles wereprepared using the corresponding feedstocks on a Büchi Mini spray dryerwith process conditions similar to those used to spray dry FormulationsI-B and I-C in Example 1, with the exception of the following processconditions. The liquid feedstock flowrate was set at 8.3 mL/min forFormulation II-B and 7 mL/min for Formulation II-C. The outlettemperature was about 83° C. for Formulation II-B and about 92° C. forFormulation II-C. The aspirator was set at 80% for Formulation II-B.

TABLE 9 Summary of liquid feedstock preparations of four batches ofparticles for Formulation II. Formulation: II-A II-B II-C II-D Liquidfeedstock mixing Static Batch Batch Static mixed mixed mixed mixed Totalsolids concentration 10 g/L 5 g/L 5 g/L 15 g/L Total solids 400 g 2.5 g9.5 g 185 g Total volume water 40 L 0.5 L 1.9 L 37 L Amount leucine in 1L 1.00 g 0.5 g 0.5 g 0.5 g Amount sodium sulfate 5.04 g 2.52 g 2.52 g2.52 g in 1 L Amount calcium chloride 5.25 g 2.61 g 2.61 g 2.61 gdihydrate in 1 L

The physical properties of the particles obtained in four separatebatches (Formulation II-A, II-B, II-C and II-D) are summarized in Table10. In addition to the data provided in Table 10, further data about thedry powders prepared from feedstock Formulation 11-A is summarized asfollows. The fine particle fraction (FPF) as measured by a full 8-stageAndersen Cascade Impactor with gravimetric analysis was on average 68.7%for FPF less than 5.6 microns and 51.5% for FPF less than 3.4 microns.The aerodynamic diameter was also measured with a full-stage ACI withgravimetric analysis. The average value for the mass median aerodynamicdiameter (MMAD) was 2.59 microns. The volume size was determined bylaser diffraction on the HELOS/RODOS sizing equipment and the averagevalue for the volume median diameter (×50) at a pressure of 1 bar was2.50 microns. In addition, the powder displayed relatively flowrateindependent behavior as can be seen from the ratio of ×50 measured at0.5 bar to ×50 measured at 4.0 bar, which was 1.47. The value for 1/4bar for these particles was 1.42.

Additional properties of the dry powders prepared by feedstockFormulation II-D are summarized as follows. The fine particle fraction(FPF) as measured by a full 8-stage Andersen Cascade Impactor withgravimetric analysis was on average 77.9% for FPF less than 5.6 micronsand 68.3% for FPF less than 3.4 microns. The aerodynamic diameter wasalso measured with a full-stage ACI with gravimetric analysis. Theaverage value for the mass median aerodynamic diameter (MMAD) was 2.17microns. The volume size was determined by laser diffraction on theHELOS/RODOS sizing equipment and the average value for the volume mediandiameter (×50) at a pressure of 1 bar was 1.90 microns. In addition, thepowder displayed relatively flowrate independent behavior as can be seenfrom the ratio of ×50 measured at 0.5 bar to ×50 measured at 4.0 bar,which was 1.17. The value for 1/4 bar for these particles was 1.63.

TABLE 10 Summary of ACI_2 data for the four batches of particles forFormulation II. Formulation: II-A II-B II-C II-D FPF less than 5.6 μm onACI-2 (%) 82.7 62.0 69.0 82.8 FPF less than 3.4 μm on ACI-2 (%) 60.147.4 53.2 70.9

Additional information relating to properties of the Formulation Hpowders and/or particles prepared in this example is provided in theTables or graphs shown in FIGS. 1A-1F and 2-4. SEM was performed asdescribed above (FIG. 5C)

Example 4

This example describes the dose emission of powders of formulationbatches I-B, II-B, and III-B from a dry powder inhaler at room andelevated conditions.

Method: Spray dried powders of the three different formulations (I-B,II-B, and III-B) were filled into size 2 HPMC capsules (Quali-V,Qualicaps, Whitsett, N.C.) to approximately half full (13-30 mgdepending on powder). Capsules were punctured prior to loading into oneof four capsule DPIs in order to ensure adequate hole openings in thecapsule. The capsules were loaded horizontally into the inhalers whichwere then connected to the custom chamber. Each dry powder inhaler had apressure transducer connected to it to monitor the flow rate through theinhaler during the test. When the test was begun, an airflow of 45 L/minwas drawn through each inhaler for 3 short bursts of 0.3 seconds each,separated by 1 minute. During each burst, the air drawn through theinhaler caused the capsule to spin and emit the powder in it into one of4 sub-chambers which had one row of 3 tissue culture wells forming thefloor of the sub-chamber. The aerosol cloud was allowed to settle forone minute before the next subsequent burst for a total of 3 bursts anda total air volume of 0.68 L being drawn through the inhaler. Theduration and total airflow rate was controlled with a flow controller(TPK-2000, MSP Corporation, Shoreview, Minn.) and recorded with an airmass flow meter (model#3063, TSI Inc., Shoreview, Minn.). Individualinhaler airflow rates were monitored with pressure sensors (model#ASCX01DN, Honeywell International Inc., Morristown, N.J.) which hadbeen previously calibrated and whose signal was converted to flow ratevia a custom Lab-view code. In one case, the custom chamber was locatedon the lab bench at room conditions, while in another 2 cases it waslocated in a stability chamber (Darwin Chambers Company, St. Louis, Mo.)set to 37° C. and 90% RH. For the first case in the stability chamber,the capsules were punctured and loaded into inhalers at room conditions,the door of the chamber was opened, the inhalers attached and the flowrate was actuated ˜30 seconds after the capsules entered the chamber. Inthe second case, the capsules were first placed unpunctured in thestability chamber for 3 minutes, then removed from the chamber,punctured and loaded at room conditions, attached in the chamber andactuated within 30 seconds of the second entry into the chamber.Following each test, the capsules were removed from the inhalers andweighed and used to calculate the percentage of powder emitted from thecapsule. For each of the 3 sets of conditions, two 12 well tissueculture plates (each plate required 4 capsules in 4 inhalers deliveringpowder to 3 wells each) were exposed to powder for each of the powderformulations tested, giving a total of 8 capsule emissions for eachpowder at each temperature and humidity setting.

As shown in Table 11 below, for all three powder hatches (I-B, II-B, andIII-B) the average amount of powder emitted from the capsule is greaterthan 99% based on the weight change of the capsule.

TABLE 11 Emitted Dose Percent Powder Batch Emitted Dose % I-B 99.45III-B 100.0 II-B 99.38

Example 5

This example describes the dispersion properties and density propertiesof formulations I-A, II-A, III-A, and Leucine formulation for placebo assummarized in Table 12. All the data found in Table 12 can also be foundin FIGS. 1A through 1E. As evidenced by the results shown in Table 12,all formulations are highly dispersible, meaning that their measuredvolume sizes are relatively independent of pressure on the HELOS/RODOS.As shown in Table 12, the ratio of the volume median sizes obtained atlow dispersion pressures (0.5 bar or 1.0 bar) and at a high dispersionpressure (4.0 bar) can be used as an indicator of dispersibility. Thesevalues are referred to as the 0.5 bar/4.0 bar ratio or the 1.0 bar/4.0bar ratio.

The tap density was determined by the modified USP<616> method using a1.5 cc microcentrifuge tube and the average value for tap density at1,000 taps were 0.29, 0.69, 0.34, and 0.04 g/cc, respectively. The MMAD,as measured by a full-stage (eight-stage) Andersen Cascade Impactor(ACI), were 2.72, 2.89, 2.59, and 4.29 um, respectively. The FPF below3.4 um, as measured on a full-stage ACI, were 41.7%, 39.7%, 51.5%, and17.4%, respectively, and below 5.6 um were 56.2%, 55.3%, 68.7%, and32.5%, respectively. The volume size was determined by laser diffractionand the average values for the volume median diameter (×50) at apressure of 1 bar were 2.57 microns, 1.51 microns, 2.50 microns, and6.47 microns, respectively. Values for pressure values at 0.5 bar, 2.0bar, and 4.0 bar can be seen in Table 12. In addition, the powderdisplayed relatively flowrate independent behavior as can be seen fromthe ratio of ×50 measured at 0.5 bar to ×50 measured at 4.0 bar as shownin Table 12. The values are 1.19, 1.12, 1.47, and 1.62, respectively.The table also includes values for the ratio of 1.0 bar to 4.0 bar, forthe sake of comparison to other art, since this is another measure offlowrate dependency.

TABLE 12 Dispersion and Density Properties of Formulations I-A, II-A,III-A Density Tap ACI-8, Gravimetric Spraytec HELOS/RODOS density MMADFPF_TD FPF_TD Dv50 Regulator x50 (g/cc) (um) <3.4 um <5.6 um (um)pressure (μm) Formulation Ave Ave Ave Ave Ave (bar) Ave 0.5 bar/4 bar 1bar/4 bar Formulation 0.29 2.72 41.7% 56.2% 3.07 0.5 2.62 1.19 1.17 I-A1.0 2.57 2.0 2.49 4.0 2.20 Formulation 0.69 2.89 39.7% 55.3% 1.78 0.51.57 1.12 1.08 III-A 1.0 1.51 2.0 1.47 4.0 1.40 Formulation 0.34 2.5951.5% 68.7% 3.05 0.5 2.59 1.47 1.42 II-A 1.0 2.50 2.0 2.17 4.0 1.76Placebo 0.04 4.29 17.4% 32.5% 21.77 0.5 7.68 1.62 1.37 (100% 1.0 6.47leucine) 2.0 5.69 4.0 4.74

Example 6

This example describes the preparation of dry powders using feedstockFormulations 6.1-6.9 as listed in Table 13 below.

TABLE 13 Feedstock Formulations 6.1-6.9 Formulation Composition andWeight % (w/w) 6.1 10.0% leucine, 58.6% calcium lactate, 31.4% sodiumchloride 6.2 50.0% leucine, 48.4% calcium lactate, 1.6% sodium chloride6.3 10.0% leucine, 66.6% calcium lactate, 23.4% sodium chloride 6.410.0% leucine, 35.1% calcium chloride, 54.9% sodium citrate 6.5 67.1%leucine, 30.0% calcium chloride, 2.9% sodium citrate 6.6 39.0% calciumchloride, 61.0% sodium citrate 6.7 10.0% leucine, 39.6% calciumchloride, 50.4% sodium sulfate 6.8 67.6% leucine, 30.0% calciumchloride, 2.4% sodium sulfate 6.9 44.0% calcium chloride, 56.0% sodiumsulfate

The general mode of preparation of the dry powders in this example issimilar to what was described for the powders in the above examples withthe exception that all of the dry powders in this example were spraydried using a Büchi B-290 spray dryer with High Performance cyclone.Formulations 6.1, 6.4, and 6.7 in this Example correspond toFormulations III-B, I-B, and II-B in the Examples above, respectively.

The physical properties of the powders and/or particles obtained in thisexample are summarized in the Tables shown in FIGS. 6A and 6B.Formulations 6.1-6.9 in Table 13 correspond to Formulations 6.1-6.9 inFIGS. 6A and 6B, respectively. In FIG. 6A, ×50 and Dv50 refer to volumemedian diameter or volume median geometric diameter (VMGD); and GSDrefers to geometric standard deviation. In FIG. 6B, yield % refers topercentage of the weight of the recovered product in the collection jarattached to the High Performance cyclone divided by the weight of thesolutes in the feedstock. All other abbreviations are describedelsewhere in the application.

Example 7

This example describes the dose emission of powders prepared byfeedstock Formulations 6.1-6.9 from a dry powder inhaler at room andelevated conditions. Some of this data is also presented above, inExample 4.

Method: Spray dried powders of the nine feedstock formulations 6.1-6.9were separately filled into size 2 HPMC capsules (Quali-V, Qualicaps,Whitsett, N.C.) to approximately half full (13-30 mg depending onpowder). Capsules were punctured prior to loading into one of fourcapsule based DPIs in order to ensure adequate hole openings in thecapsule. The capsules were loaded horizontally into the inhalers whichwere then connected to the custom chamber. Each dry powder inhaler had apressure transducer connected to it to monitor the flow rate through theinhaler during the test. When the test was begun, an airflow of 45 L/minwas drawn through each inhaler for 3 short bursts of 0.3 seconds each,separated by 1 minute. During each burst, the air drawn through theinhaler caused the capsule to spin and emit the powder in it into one of4 sub-chambers which had one row of 3 tissue culture wells forming thefloor of the sub-chamber. The aerosol cloud was allowed to settle forone minute before the next subsequent burst for a total of 3 bursts anda total air volume of 0.68 L being drawn through the inhaler. Theduration and total airflow rate was controlled with a flow controller(TPK-2000, MSP Corporation, Shoreview, Minn.) and recorded with an airmass flow meter (model#3063, TSI Inc., Shoreview, Minn.). Individualinhaler airflow rates were monitored with pressure sensors (model#ASCX01DN, Honeywell International Inc., Morristown, N.J.) which hadbeen previously calibrated and whose signal was converted to flow ratevia a custom Lab-view code. In one case, the custom chamber was locatedon the lab bench at room conditions, while in another 2 cases it waslocated in a stability chamber (Darwin Chambers Company, St. Louis, Mo.)set to 37° C. and 90% RH. For the first case in the stability chamber,the capsules were punctured and loaded into inhalers at room conditions,the door of the chamber was opened, the inhalers attached and the flowrate was actuated ˜30 seconds after the capsules entered the chamber. Inthe second case, the capsules were first placed unpunctured in thestability chamber for 3 minutes, then removed from the chamber,punctured and loaded at room conditions, attached in the chamber andactuated within 30 seconds of the second entry into the chamber.Following each test, the capsules were removed from the inhalers andweighed and used to calculate the percentage of powder emitted from thecapsule. For each of the 3 sets of conditions, two 12 well tissueculture plates (each plate required 4 capsules in 4 inhalers deliveringpowder to 3 wells each) were exposed to powder for each of the powderformulations tested, giving a total of 8 capsule emissions for eachpowder at each temperature and humidity setting.

As shown in Table 14 below, for all nine powder batches (obtained usingfeedstock Formulations 6.1-6.9) the average amount of powder emittedfrom the capsule is greater than 98% based on the weight change of thecapsule.

TABLE 14 Emitted Dose Percent Formulation Emitted Dose (%) 6.1 100.00%6.2 98.86% 6.3 99.85% 6.4 99.45% 6.5 99.68% 6.6 100.00% 6.7 99.38% 6.898.05% 6.9 100.00%

Example 8

This example describes the results of a short-term stability study thatwas conducted for the dry powders prepared by feedstock Formulations6.1, 6.4 and 6.7.

An important characteristic of pharmaceutical dry powders is stabilityat different temperature and humidity conditions. One property that maylead to an unstable powder is the powder's tendency to absorb moisturefrom the environment, which then will likely lead to agglomeration ofthe particles, thus altering the apparent particle size of the powder atsimilar dispersion conditions. Spray dried powders were held at a rangeof conditions for periods of one week to three or more months andperiodically tested for particle size distribution. Storage conditionsincluded closed capsules in vials at 25° C. and 60% RH, closed capsulesin vials at 40° C. and 75% RH, closed capsules at room temperature and40% RH, open capsules at 30° C. and 65% RH and open capsules at 30° C.and 75% RH. Size 3 HPMC capsules (Quali-V, Qualicaps, Whitsett, N.C.)were half filled with each dry powder. One sample was tested immediatelyin the Spraytec (Malvern Instruments Inc., Westborough, Mass.), a laserdiffraction spray particle sizing system where dry powders can bedispersed from an inhaler using the inhaler cell setup. Approximately 16capsules were filled with each powder prepared using feedstock solutions6.1, 6.4 and 6.7. Capsules were kept in the lab at controlled humidityand temperature conditions (˜23-28% RH), and also in the outside lab atvarying temperature and relative humidity (˜40-75% RH). Capsules kept atstorage conditions of 25° C. and 60% RH, 40° C. and 75% RH, 30° C. and65% RH and 30° C. and 75% RH were held in stability chambers (DarwinChambers Company, St. Louis, Mo.) set at those conditions. At specifictime points (ranging from 30 min to 3 months), one to three capsulesfrom each condition were tested on the Spraytec for geometric particlesize distribution and the ACI-2 for aerodynamic particle sizeproperties.

Generally, the powders that were in closed capsules in vials remainedstable for a long period of time, longer than three months. Powders thatwere in open capsules with no vials showed agglomeration after exposureto higher humidity conditions. The stability data are summarized inTable 15 below.

TABLE 15 Short-term Stability Data closed capsules closed capsules, opencapsules, no vials in vials no vials Spraytec ACI-2 Spraytec ACI-2Counter 25 C./60% 40 C./75% Spraytec ACI-2 30 C./65% 30 C./65% 30 C./75%30 C./75% Formulation ion Excipient RH RH 40% RH 40% RH RH RH RH RH 6.1Lactate 10% >3 0.5-1 >8 4-6 >30 >30 >30 >30 Leucine months month daysdays min min min min 6.4 Citrate 10% >3 1-3 >7 N/A >30 >30 <30 >30Leucine months months days min min min min 6.7 Sulfate 10% >3 1-3 2-7N/A >30 >30 >30 >30 Leucine months months days min min min min

Example 9

This example describes a Bacterial Pass-Through Assay performed usingdry powders prepared using feedstock Formulations A-E found in Table 16.

Method: To test the effect of aerosolized dry powder formulations onbacterial movement across mucus, a pass-through model was used. In thismodel, 200 μL of 4% sodium alginate (Sigma-Aldrich, St. Louis, Mo.) wasadded to the apical surface of a 12 mm Costar Transwell membrane(Corning, Lowell, Mass.; 3.0 μm pore size) and subsequently exposed todry powder formulations. Dry powders were aerosolized into the chamberusing a dry powder insufflator (Penn-Century, Inc., Philadelphia, Pa.)and allowed to settle by gravity over a 5 minute period. Following thisexposure, 10 μL of Klebsiella pneumoniae (˜10⁷ CFU/mL in saline) wasadded to the apical surface of the mimetic. At various time points afterthe addition of bacteria, aliquots of the basolateral buffer wereremoved and the number of bacteria in each aliquot was determined byserially diluting and plating on blood agar plates. A schematic of thismethod is shown in FIG. 7. The concentration of salt that was deliveredto each Transwell was quantified by HPLC. For this purpose, empty wellsof the 12 well cell culture plate that were next to each Transwell andwere exposed to the same dose of formulation were rinsed with sterilewater and diluted 1:1 with acetic acid to solubilize the calcium saltsin each powder.

The effect of calcium containing powders on K. pneumoniae movementthrough sodium alginate mucus mimetic was tested. Dry powderformulations comprising calcium salts with different solubilityprofiles, together with leucine and sodium chloride, were screened foractivity. Table 16 (below) lists the feedstock formulations of thepowders that were tested. A 50.0% (w/w) leucine loading in thecomposition was necessary, as opposed to the 10.0% (w/w) leucine loadingin the formulations described in the examples above, due to dosing anddetection limitations in the pass through model. The calcium and sodiummolar ratio was chosen for each formulation to target a 1:1 molar ratio,while not needing to go too low on the relative weights of anyparticular salt. Therefore, the lactate, citrate, and acetateformulations used were not in a 1:1 molar ratio in order to keep theweights of the sodium chloride and the calcium chloride in thoseformulations, respectively, above about 10% by weight.

TABLE 16 Feedstock Formulations Ca:Na mole Formulation Composition (w/w)ratio A 50.0% leucine, 22.0% 1.0:2.0 calcium chloride, 28.0% sodiumsulfate B 50.0% leucine, 25.5% 1.0:2.0 calcium chloride, 24.5% sodiumcarbonate C 50.0% leucine, 19.5% 1.0:2.0 calcium chloride, 30.5% sodiumcitrate D 50.0% leucine, 37.0% 1.0:1.3 calcium lactate, 13.0% sodiumchloride E 50.0% leucine, 33.75% 1.0:1.8 calcium acetate, 16.25% sodiumchloride

The results for this test are shown in FIGS. 8A and 8B. The twodifferent figures represent two different sets of experiments, run atthe same conditions. The leucine control and sulfate data allow forrelative comparison between the two sets of experiments. The powderscontaining the anions sulfate, lactate, and acetate, i.e., the drypowders prepared from feedstock formulations A, D, and E, respectively,reduced the movement of bacteria across the mimetic, whereas the powderscontaining the anions carbonate and citrate, i.e., dry powders preparedfrom feedstock formulations B and C, exhibited no effect. These findingcorrelated with the known solubility of the calcium salts in water,suggesting that the possible failure of carbonate and citrate salts toinhibit the movement of K. pneumoniae could be related to the solubilityof these powders at the surface of the sodium alginate mimetic. Thisconclusion is also based on the plausible assumption that the ionexchange reaction described previously goes to completion during spraydrying, and that the form of the calcium salt in Formulations A throughE is calcium sulfate, calcium carbonate, calcium citrate, calciumlactate, and calcium acetate, respectively. The solubility of thesesalts from least soluble to most soluble: calcium carbonate<calciumcitrate<calcium sulfate<calcium lactate<calcium acetate. (See Table 1above.)

Example 10

This example describes the performance of dry powders in reducing viralreplication utilizing a viral replication model.

In this example, a series of dose response studies with different drypowder prepared from feedstock formulations consisting of differentcalcium salts are described. Dry powders were made with leucine, acalcium salt (lactate or chloride), and sodium salt (chloride, sulfate,citrate or carbonate). Feedstock formulations listed 10-1, 10-2 and 10-3were spray dried on a Büchi B-290 mini spray dryer. The system used theBüchi B-296 dehumidifier to ensure stable temperature and humidity ofthe air used to spray dry. Feedstock Formulation 10-4 was spray dried ona Niro Mobile Minor Spray Dryer in an open cycle with nitrogen.

Four liquid feedstocks were prepared with the following components andratios (weight percentage) as listed in Table 17.

TABLE 17 Feedstock Formulations Feedstock Composition Ca:Na moleFormulation (w/w) ratio 10-1 50.0% leucine, 37.0% 1.0:1.3 calciumlactate, 13.0% sodium chloride 10-2 50.0% leucine, 22.0% 1.0:2.0 calciumchloride, 28.0% sodium sulfate 10-3 50.0% leucine, 19.5% 1.0:2.0 calciumchloride, 30.5% sodium citrate 10-4 50.0% leucine, 25.5% 1.0:2.0 calciumchloride, 24.5% sodium carbonate

A 50.0% (w/w) leucine loading in the composition was necessary, asopposed to the 10.0% (w/w) leucine loading in the formulations describedin the examples above, due to dosing and detection limitations in theviral replication model. The calcium and sodium mole ratio was chosenfor each formulation to target a 1:1 molar ratio, while not needing togo too low on the relative weights of any particular salt. Therefore,the lactate and citrate formulations used were not in a 1:1 mole ratioin order to keep the weights of the sodium chloride and the calciumchloride in those formulations, respectively, above about 10% by weight.

Formulations 10-1, 10-2 and 10-3 were spray dried with feedstock solidsconcentrations of 5 g/L, while the exact amount of salts and excipientdissolved in ultrapure water and its specific volume varied. Thefollowing process settings were used: inlet temperature of 220° C.,liquid flow rate of approximately 10mL/min, room conditions at23.2-24.6° C. and 19-21% RH, and dehumidifier air at 3-5° C. and 30% RH.The outlet temperature, cyclone and aspirator rate varied. Formulation10-1 was spray dried using a high performance cyclone with the aspiratorat 80% and an outlet temperature of 93° C. Dry powder formulations 10-2and 10-3 were made with the regular cyclone, an aspirator at 100% and anoutlet temperature of 111-115° C. Formulation 10-4 was spray dried witha solids concentration of 2.7 g/L and the following process settings:inlet temperature of 140° C., outlet temperature of 75° C., liquidfeedstock flowrate of 30 ml/min, process gas flowrate of 100 kg/hr,atomizer gas flowrate of 20 g/min and a spray drying drum chamberpressure of −2 “WC.

A cell culture model of Influenza infection was used to study theeffects of Formulations 1 through 4. Calu-3 cells (American Type CultureCollection, Manassas, Va.) were cultured on permeable membranes (12 mmTranswells; 0.4 μm pore size, Corning Lowell, Mass.) until confluent(the membrane was fully covered with cells) and air-liquid interface(ALI) cultures were established by removing the apical media andculturing at 37° C./5% CO₂. Cells were cultured for >2 weeks at ALIbefore each experiment. Prior to each experiment the apical surface ofeach Transwell was washed 3× with PBS (Hyclone, Logan, Utah). Calu-3cells were exposed to dry powders using a proprietary dry powdersedimentation chamber. In order to expose cells to equivalent doses ofcalcium, capsules were filled with different amounts of each powder. Thehigh, medium, and low fill weights were calculated based on matching theamount of calcium delivered by each powder (4.23 mg, 1.06 mg, and 0.35mg). For each dry powder condition tested, two capsules were weighed asempty, filled, and after exposure in order to determine emitted dose ofthe powder. Table 18 (below) shows the capsule fill weights before andafter exposure and the concentration of calcium delivered to cells asdetermined by HPLC measurements. Immediately after exposure, thebasolateral media (media on the bottom side of the Transwell) wasreplaced with fresh media. Triplicate wells were exposed to dry powdersfrom each feedstock formulation in each test. A second cell cultureplate was exposed to the same dry powders from the feedstockformulations to quantify the delivery of total salt or calcium to cells.One hour after exposure, cells were infected with 104 of InfluenzaA/WSN/33/1 (H1N1) or Influenza A/Panama/2007/99 (H3N2) at a multiplicityof infection of 0.1-0.01 (0.1-0.01 virions per cell). Four hours afteraerosol treatment, the apical surfaces were washed to remove excess drypowders and unattached virus and cells were cultured for an additional20 h at 37° C. plus 5% CO₂. Twenty-four hours after aerosol treatment,virus released onto the apical surface of infected cells was collectedin culture media or PBS and the concentration of virus in the apicalwash was quantified by TCID₅₀ (50% Tissue Culture Infectious Dose)assay. The TCID₅₀ assay is a standard endpoint dilution assay that isused to quantify how much of a virus is present in a sample.

Dry powder formulations were tested to evaluate their effect onInfluenza A/WSN/33/1 infection in a cell culture model (Table 18). Todeliver an equivalent amount of calcium ion Ca²⁺, the desired fillweight was calculated for each dry powder formulation. Qualicap capsuleswere weighed empty, filled, and after exposure to determine the emitteddose. Triplicate wells were exposed to each capsule and after wells werewashed. HPLC analysis of these samples determined the amount of Ca²⁺delivered to cells. * denotes the use of two capsules in order toachieve desired fill weight. ^(a) denotes n=3, ^(b) denotes n=1

TABLE 18 Dry powder, prepared from feedstock formulations 10-1 to 10-4,tested to evaluate their effect on Influenza A/WSN/33/1 infection in acell culture model. Feedstock Calcium ion Formula- Capsule concentrationtion Intended Empty Filled after determined (for Dry Fill CapsuleCapsule Exposure by HPLC Powders) (mg) (mg) (mg) (mg) (μg/cm²) 10-253.18 31.7 83.0 31.9 20.5 ± 0.7^(a) (50.0% 13.29 32.5 45.9 33.9 5.8^(b)leucine, 4.43 33.3 38.4 33.9 2.8^(b) 22.0% calcium chloride, 28.0%sodium sulfate) 10-1 62.17 64.972, 99.649, 64.994, 50.9 ± 1.1^(a) (50.0%63.122* 98.881* 63.679* leucine, 15.54 63.525 81.926 68.141 12.7 ±1.7^(a) 37.0% 5.18 62.453 67.796 62.49 4.0^(b) calcium lactate, 13.0%sodium chloride) 10-3 60.0 64.4 123.6 81.994 20.5 ± 5.7^(a) (50.0% 14.9964.0 78.5 65.388  7.6 ± 0.9^(a) leucine, 5.00 63.5 70.3 63.829  3.6 ±1.5^(a) 19.5% calcium chloride, 30.5% sodium citrate) 10-4 45.88 64.6104.7 66.685 28.1 ± 7.3^(a) (50.0% 11.47 61.5 72.0 63.186  8.1 ± 2.6^(a)leucine, 3.82 61.8 62.6 63.341 5.62 ± 2.7^(a) 25.5% calcium chloride,24.5% sodium carbonate)

Example 10A

Dry powders, prepared from feedstock formulations 10-1 to 10-4, reduceInfluenza A/WSN/33/1 (H1N1) infection in a dose-dependent manner.

To test the effect of dry powder formulations on Influenza infection ina cell culture model Calu-3 cells were exposed to four different drypowder formulations each consisting of 50% leucine, a calcium salt andsodium chloride. Viral infection was assessed by quantifying the amountof viral replication over a 24 h period. The specific powders tested arelisted in Table 18 (above), and included carbonate, lactate, sulfate andcitrate salts. In an attempt to expose cells to equivalent amounts ofcalcium of each of the four calcium containing powders, capsules werefilled to appropriate fill weights prior to dosing. Cells exposed to noformulation (Air) were used as control cells.

As seen in FIG. 9, each powder exhibited a dose-responsive reduction ininfluenza infection; however, the magnitude of the effect was differentamong the four powders tested. At low calcium concentrations calciumlactate was most efficacious suggesting that it was the most potent ofthe powders tested. At higher concentrations of calcium, the calciumlactate and calcium citrate powders exhibited similar efficacy.Additional testing of the calcium citrate powder at even higherconcentrations may demonstrate that it is the most efficacious powder.The calcium sulfate powder exhibited an intermediate effect and wascomparable to calcium citrate at several concentrations. Calciumcarbonate had only a minimal effect on viral replication even at thehighest concentration (less than 10-fold). Of note, calcium carbonate isthe least soluble of the powders tested.

As shown in FIG. 9, the dry powders prepared for this reduce Influenzainfection in a dose-dependent manner. Calu-3 cells exposed to noformulation were used as a control and compared to Calu-3 cells exposedto dry powder formulations at different fill weights. The concentrationof virus released by cells exposed to each aerosol formulation wasquantified. Bars represent the mean and standard deviation of triplicatewells for each condition. Data were analyzed statistically by one wayANOVA and Tukey's multiple comparison post-test.

Example 10B

Dry powder, prepared from feedstock formulations 10-1 to 10-4 in Table19, reduce Influenza A/Panama/2007/99 (H3N2) infection in adose-dependent manner.

To extend these studies, the same powders were tested with a secondinfluenza strain [Influenza A/Panama/2007/99 (H3N2)]. Similar to Example10A, Calu-3 cells were exposed to four different dry powder formulationseach consisting of 50% leucine, a calcium salt and sodium chloride.Viral infection was assessed by quantifying the amount of viralreplication over a 24 h period. The specific powders tested are listedin Table 19 (below) and included carbonate, lactate, sulfate and citratesalts. In an attempt to expose cells to equivalent amounts of calcium ofeach of the four calcium containing powders, capsules were filled toappropriate fill weights prior to dosing. Cells exposed to noformulation (Air) were used as control cells.

As seen in FIG. 10, using this strain, similar efficacy was observed foreach powder: calcium lactate was the most efficacious, calcium citrateand calcium sulfate exhibited intermediate efficacy and the calciumcarbonate powder was only minimally efficacious. These data support thebroad activity of Ca:Na dry powders against multiple influenza strains.

Dry powders, prepared from feedstock formulations 10-1 to 10-4, testedto evaluate their effect on Influenza A/Panama/99/2007 (II3N2) infectionin a cell culture model (Table 19). To deliver equivalent amount ofCa²⁺, the desired fill weight was calculated for each dry powderformulation. Qualicap capsules were weighed empty, filled, and afterexposure to determine the emitted dose. Triplicate wells were exposed toeach capsule and after wells were washed. HPLC analysis of these samplesdetermined the amount of Ca²⁺ delivered to cells.

TABLE 19 Feedstock Calcium ion Formula- Capsule concentration tionDesired Empty Filled after determined (for Dry Fill Capsule CapsuleExposure by HPLC Powders) (mg) (mg) (mg) (mg) (μg/cm² ± SD)^(a) 10-253.18 61.358 121.417 62.591 40.8 ± 5.0  (50.0% 13.29 60.602 76.80462.167 10.5 ± 2.3  leucine, 22.0% 4.43 65.102 70.789 65.670 2.9 ± 0.6calcium chloride, 28.0% sodium sulfate) 10-1 62.17 64.037 125.465 67.04333.8 ± 3.5  (50.0% 15.54 65.358 82.474 65.632 9.7 ± 1.4 leucine, 37.0%5.18 66.046 72.455 66.324 3.4 ± 0.9 calcium lactate, 13.0% sodiumchloride) 10-3 60.0 62.581 108.035 63.841 29.6 ± 10.1 (50.0% 14.9963.393 75.770 64.085 8.1 ± 1.4 leucine, 19.5% 5.00 65.910 70.062 66.2044.1 ± 0.8 calcium chloride, 30.5% sodium citrate) 10-4 45.88 64.506115.876 65.004 30.4 ± 11.9 (50.0% 13.47 64.319 77.627 65.080 11.1 ± 4.3 leucine, 25.5% 3.82 66.495 71.398 66.698 2.4 ± 1.0 calcium chloride,24.5% sodium carbonate)

As shown in FIG. 10, the dry powders prepared for this Example reduceInfluenza A/Panama/99/2007 (H3N2) infection in a dose-dependent manner.Calu-3 cells exposed to no formulation (0 μg Ca²⁺/cm²) were used as acontrol and compared to Calu-3 cells exposed to dry powder formulationsat different fill weights and therefore different concentrations ofcalcium. The concentration of calcium delivered to cells in eachexperiment for each fill weight was determined using HPLC measurementsof calcium in washes from empty plates exposed to each condition. Theconcentration of virus released by cells exposed to each aerosolformulation 24 h after dosing was quantified by TCID₅₀ assay. Each datapoint represents the mean and standard deviation of triplicate wells foreach condition.

Example 11 In Vivo Influenza Model

This example demonstrates that dry powder formulations comprised ofcalcium salts and sodium chloride reduce the severity of influenzainfection in ferrets. The formulations tested are shown in Table 20.Control ferrets were exposed to a powder comprised of 100% leucine underthe same exposure conditions. In preliminary in vitro studies, thiscontrol powder had no effect on viral replication. Calcium powders andcontrol (Formulation I, Formulation II, Formulation III and Leucinecontrol) were aerosolized with a Palas Rotating Brush Generator 1000solid particle disperser (RBG, Palas GmbH, Karlsruhe, Germany). Ferrets(n=8 per group) were exposed to ˜0.2 mg Ca/kg and the severity ofinfection was evaluated over time. Each formulation was dispersed in anose-only exposure system 1 hour before infection, 4 hours afterinfection and then BID for 4 days (d1-4). The study was terminated onday 10. Body temperatures were determined twice a day beginning on day 0of the study. Ferrets infected with influenza typically show increasesin body temperature within 2 days of infection, drop body weight overthe course of the study and show clinical signs of infection such aslethargy and sneezing. These changes coincide with an increase ininfluenza viral titers shed from the nasal cavity and increases in nasalinflammation.

TABLE 20 Formulations tested for efficacy in ferrets FormulationComposition Formulation I 10.0% leucine, 35.1% calcium chloride, 54.9%sodium citrate (Active with 12.7% calcium ion) Formulation II 10.0%leucine, 39.6% calcium chloride, 50.4% sodium sulfate (Active with 14.3%calcium ion) Formulation III 10.0% leucine, 58.6% calcium lactate, 31.4%sodium chloride (Active with 10.8% calcium ion)

On study day −4, ferrets were implanted with a microchip subcutaneouslyin the right rear flank and another in the shoulder for redundancy. Thetransponder chip (IPTT-300 Implantable Programmable Temperature andIdentification Transponder; Bio Medic Data Systems, Inc, Seaford, Del.19973) allows for ferret identification and provides subcutaneous bodytemperature data throughout the study using a BMDS electronic proximityreader wand (WRS-6007; Biomedic Data Systems Inc, Seaford, Del.).Subcutaneous body temperatures taken on day −3 to −1 were used asbaseline temperatures and used to calculate the change from baseline foreach animal over the course of the study. Treatment with a dry powderformulation comprised of leucine (excipient), Ca-lactate (FormulationIII), and NaCl had a significant impact on body temperature increases(FIG. 11C). The mean body temperature changes in this group remained ator below baseline measurements for the course of the study and the areaunder the curve (AUC) measurements were approximately 5-fold lower thanthe control (FIG. 11D). The two other powders tested exhibited lesspronounced efficacy that was limited to differences from the control onspecific days of the study. In particular, both the Ca citrate and Casulfate treated groups had lower body temperatures than the controlanimals on day 3 of the study (FIGS. 11A and 11B, respectively) and theCa sulfate group had lower body temperatures over the final three daysof the study.

Example 12

This example demonstrates that dry powder formulations comprised ofdifferent excipients reduce influenza infection, but at higher dosesthan formulations comprised of leucine.

To assess the impact of the excipient on efficacy in vitro we tested twodry powder formulations (Table 21) that varied in excipient and comparedtheir efficacy to Formulation III (containing leucine) using theinfluenza replication model. These formulations contained the sameconcentration of calcium lactate and sodium chloride and the same weightpercentage of excipient (10%).

TABLE 21 Formulations used to evaluate efficacy against multipleinfluenza viruses and to test different excipients Ca:Na molar SprayFormulation Feedstock Composition (w/w) ratio Dryer I 10.0% leucine,35.1% calcium chloride, 1:2 Niro 54.9% sodium citrate (Active with 12.7%calcium ion) II 10.0% leucine, 39.6% calcium chloride, 1:2 Niro 50.4%sodium sulfate (Active with 14.3% calcium ion) III 10.0% leucine, 58.6%calcium lactate, 1:2 Niro 31.4% sodium chloride (Active with 10.8%calcium ion) V 10.0% marmitol, 58.6% calcium lactate, 1:2 Büchi 31.4%sodium chloride (Active with 10.8% calcium ion) IV 10.0% maltodextrin,58.6% calcium 1:2 Büchi lactate, 31.4% sodium chloride (Active with10.8% calcium ion)

Calu-3 cells exposed to no formulation were used as a control andcompared to Calu-3 cells exposed to dry powder comprised of calciumlactate and sodium chloride with different excipients. Three differentfill weights of the mannitol and maltodextrin powders were used to covera dose range between 10 to 30 μg Ca2+/cm2. The concentration of virusreleased by cells exposed to each aerosol formulation was quantified(FIG. 12). Each data point represents the mean and standard deviation ofduplicate wells for each concentration. Data were analyzed by one-wayANOVA and Tukey's multiple comparisons post-test. The data for the lowdose of each powder is representative of two independent experiments.

Both the mannitol and maltodextrin containing formulations reducedinfluenza infection in a dose responsive manner, however, they weresignificantly less potent than the leucine containing powder. At a doseof 14.8 μg Ca^(2+/)cm², the leucine containing powder reduced influenzainfection by 2.9±0.2 log₁₀ TCID₅₀/mL, whereas the mannitol powder at acomparable dose (12.2 μg Ca^(2+/)cm²) reduced infection by 0.85±0.0log₁₀ TCID₅₀/mL and the maltodextrin powder (11.9 μg Ca²⁺/cm²) had noeffect on replication (FIG. 12). Even at higher doses (>27 μg Ca²⁺/cm²),the maximal reduction for mannitol (1.9±0.50 log 10 TCID₅₀/mL) andmaltodextrin (2.2±0.14 log₁₀ TCID₅₀/mL) was less than that of theleucine powder. Of note, previous testing using powders comprised of100% leucine found no effect of the excipient alone on viralreplication. These data suggest that the nature of the excipient canimpact the efficacy of calcium containing formulations.

Example 13

This example demonstrates the efficacy of dry powder formulationscomprising calcium salt, calcium lactate, calcium sulfate or calciumcitrate powders with respect to treatment of influenza, parainfluenza orrhinovirus.

The Formulation I, Formulation II, and Formulation III powders wereproduced by spray drying utilizing a Mobile Minor spray dryer (Niro, GEAProcess Engineering Inc., Columbia, Md.). All solutions had a solidsconcentration of 10 g/L and were prepared with the components listed inTable 22. Leucine and calcium salt were dissolved in DI water, andleucine and sodium salt were separately dissolved in DI water with thetwo solutions maintained in separate vessels. Atomization of the liquidfeed was performed using a co-current two-fluid nozzle (Niro, GEAProcess Engineering Inc., Columbia, Md.). The liquid feed was fed usinggear pumps (Cole-Parmer Instrument Company, Vernon Hills, Ill.) into astatic mixer (Charles Ross & Son Company, Hauppauge, N.Y.) immediatelybefore introduction into the two-fluid nozzle. Nitrogen was used as thedrying gas and dry compressed air as the atomization gas feed to thetwo-fluid nozzle. The process gas inlet temperature was 282° C. andoutlet temperature was 98° C. with a liquid feedstock rate of 70 mL/min.The gas supplying the two-fluid atomizer was approximately 14.5 kg/hr.The pressure inside the drying chamber was at −2 “WC. Spray driedproduct was collected in a container from a filter device.

TABLE 22 Formulations used to evaluate efficacy against differentrespiratory viruses Ca:Na molar Spray Formulation Feedstock Composition(w/w) ratio Dryer I 10.0% leucine, 35.1% calcium chloride, 1:2 Niro54.9% sodium citrate (Active with 12.7% calcium ion) II 10.0% leucine,39.6% calcium chloride, 1:2 Niro 50.4% sodium sulfate (Active with 14.3%calcium ion) III 10.0% leucine, 58.6% calcium lactate, 1:2 Niro 31.4%sodium chloride (Active with 10.8% calcium ion)

A cell culture model of Influenza A/Panama/2007/99, human parainfluenzatype 3 (hPIV3) or Rhinovirus (Rv16) infection was used to evaluate theefficacy of dry powder formulations. This model has been described indetail previously (See, Example 10) and utilizes Calu-3 cells grown atair-liquid interface as a model of influenza infection of airwayepithelial cells. Calu-3 cells were exposed to dry powders using a drypowder sedimentation chamber. The amount of calcium ion (Ca2+) deliveredto each well was determined by HPLC using dry powder recovered from anempty well in the cell culture plate. The concentration of calciumdeposited in each study is shown in Table 23.

TABLE 23 Calcium Deposition Formulation I (μg Ca/cm²) Formulation II (μgCa/cm²) Formulation III (μg Ca/cm²) Low Medium High Low Medium High LowMedium High Influenza 12.74 17.12 28.85 11.37 15.84 27.73 10.93 16.0126.61 Parainfluenza 10.58 16.19 25.04 12.26 15.71 25.32 11.03 16.8126.33 Rhinovirus 11.63 16.25 24.11 10.86 15.01 23.89 11.49 15.22 24.69

One hour after exposure, cells were infected with 10 μL of InfluenzaA/Panama/99/2007 at a multiplicity of infection of 0.1-0.01 (0.1-0.01virions per cell), human parainfluenza type 3 (hPIV3) at a multiplicityof infection of 0.1-0.01 (0.1-0.01 virions per cell), or 10 μL ofrhinovirus (Rv16) at a multiplicity of infection of 0.1-0.01 (0.1-0.01virions per cell). Four hours after dry powder treatment, the apicalsurfaces were washed to remove excess formulation and unattached virus,and cells were cultured for an additional 20 hours at 37° C. plus 5%CO₂. The next day (24 hours after infection) virus released onto theapical surface of infected cells was collected in culture media and theconcentration of virus in the apical wash was quantified by TCID₅₀ (50%Tissue Culture Infectious Dose) assay. The TCID₅₀ assay is a standardendpoint dilution assay that is used to quantify how much of a givenvirus is present in a sample. For each of the three powders, Calu-3cells were exposed to three different Ca²⁺ doses and the replication ofeach virus was assessed.

Influenza

In the influenza model, all three powders significantly reduce viraltiter to comparable levels at the highest dose tested: Formulation I,Formulation II, and Formulation III reduced viral titer up to 3.25,3.80, and 3.95 log₁₀ TCID₅₀/mL, respectively (FIG. 13A). It is importantto note that while at the highest dose tested these powders exhibitedsimilar activity against influenza, at lower doses the data suggests themost efficacious powder was Formulation III (comprised of leucine,calcium lactate and sodium chloride). Formulation III reduced viraltiters 3.70 and 3.75 log TCID₅₀/mL at low and medium doses, whereas lowdoses of Formulation I and Formulation II reduced viral titer 2.50 and2.95 log₁₀ TCID₅₀/mL, and mid doses of Formulation I and Formulation IIreduced viral titers 2.65 and 3.30 log₁₀ TCID₅₀/mL, respectively.

Parainfluenza

Formulation I, Formulation II, and Formulation III were tested over asimilar dose range against parainfluenza. The parainfluenza titer in theFormulation II treated cell cultures was comparable to the control cells(FIG. 13B) at doses of calcium similar to those used in the influenzaexperiment, indicating that the calcium sulfate based formulation mayexhibit activity only against specific pathogens. In contrast,Formulation I and Formulation III treatment resulted in a dose dependentreduction in parainfluenza infection. At high doses, Formulation I andFormulation III reduced infection by 2.70 and 4.10 log₁₀ TCID₅₀/mL,respectively, compared to the control cells. Similarly, Formulation IIIexhibited greater efficacy than Formulation I at the middle dose tested,however, neither formulation reduced infection at the lowest dose tested(FIG. 13B; Table 25). Collectively, these data demonstrate that calciumbased dry powder formulations effectively reduce the infectivity ofparainfluenza. These effects are specific to certain calcium salts andthe efficacious dose ranges differ significantly from that observed forinfluenza.

Rhinovirus

Influenza and parainfluenza are enveloped viruses. To test the broadspectrum activity of calcium dry powder formulations and extend thesefindings to nonenveloped viruses, the same powders were tested againstrhinovirus. All three formulations reduced rhinovirus to some extent,with the Formulation III powder demonstrating the greatest activity(FIG. 13C). Formulation III treatment resulted in a significant, 2.80log₁₀ TCID₅₀/mL viral reduction at the highest dose tested. Low andmedium doses of this powder reduced titer 1.15 and 2.10 log₁₀ TCID₅₀/mL,respectively, compared to control cells. Formulation I and FormulationII treatment also reduced rhinovirus infection, albiet to a lesserextent than Formulation III. At the highest dose tested, Formulation Ireduced infection by 1.70 log₁₀ TCID₅₀/mL and Formulation II reducedinfection 1.60 log₁₀ TCID₅₀/mL. Together these results indicate thatcalcium based dry powder formulations can be broadly applied to diverseviral infections.

The above data suggests that by increasing the delivered dose of calciumdry powder formulations exhibit more activity than was previouslyobserved at lower doses. Influenza infection was reduced by all threepowders tested, although the calcium lactate based formulation(Formulation III) exhibited greater potency than the calcium sulfate(Formulation II) and calcium citrate (Formulation I) formulations.Additionally, across all three viral strains, Formulation III treatmentresulted in the greatest reduction in viral titer. At higher dosesFormulation I effectively reduced viral titer in all three viralstrains, but the effect was much more pronounced with influenza andparainfluenza, suggesting a difference in mechanism that may be relatedto viral strain specificity. Formulation II treatment was active againstparainfluenza, but exhibited better activity against both influenza andrhinovirus, suggesting that the specific calcium counterions may havesome role in the optimal activity of the formulation.

Example 14 Calcium Lactate, Sodium Chloride, Maltodextrin Dry Powder

This example describes the preparation of dry powders using feedstock ofFormulation IV: 10.0 weight percent maltodextrin, 58.6 weight percentcalcium lactate and 31.4 weight percent sodium chloride.

An aqueous phase was prepared for a batch process by dissolvingmaltodextrin in ultrapure water, then calcium lactate pentahydrate, andfinally sodium chloride. The solution was kept agitated throughout theprocess until the materials were completely dissolved in the water atroom temperature. For the maltodextrin and calcium lactate formulation,three batches (A, B & C) of feedstock were prepared and spray dried.Details on the liquid feedstock preparations for each of the threehatches are shown in Table 24, where the total solids concentration isreported as the total of the dissolved anhydrous material weights. Thesolutions or suspensions were then spray dried using a BUN spray dryer.For each formulation, three batches (A, B & C) of feedstock wereprepared and spray dried. Batch A, B and C particles were prepared usingthe corresponding feedstocks on a Büchi Mini spray dryer with processconditions similar to those used to spray dry for Formulations I-B andI-C in Example 1, with the exception of the following processconditions. The liquid feedstock flow rate was set at 5.2 mL/min forFormulation IV-A and Formulation IV-B and 5.6 mL/min for FormulationIV-C. The outlet temperature was about 90° C. to 98° C. for FormulationIV-A, about 100° C. to for Formulation IV-B and about 100° C. 106° C.for Formulation IV-C.

TABLE 24 Summary of liquid feedstock preparations of three batches ofparticles for Formulation IV. Formulation: IV-A IV-B IV-C Liquidfeedstock mixing Batch Batch Batch mixed mixed mixed Total solidsconcentration 5 g/L 5 g/L 5 g/L Total solids 5 g 5 g 20 g Total volumewater 1.0 L 1.0 L 4.0 L Amount leucine in 1 L 0.5 g 0.5 g 0.5 g Amountsodium chloride in 1.55 g 1.55 g 1.55 g 1 L Amount calcium lactate 4.13g 4.13 g 4.13 g pentahydrate in 1 L

Some of the physical properties of the particles obtained in threeseparate batches (Formulation IV-A, IV-B, and IV-C) are summarized inTable 25. In addition to the data provided in Table 25, further dataabout the dry particles prepared by feedstock formulation IV-A issummarized as follows. The fine particle fraction (FPF) as measured by acollapsed 2-stage Andersen Cascade Impactor with gravimetric analysiswas on average 71.3% for FPF less than 5.6 microns and 47.5% for FPFless than 3.4 microns. The volume size was determined by laserdiffraction on the HELOS/RODOS sizing equipment and the average valuefor the volume median diameter (×50) at a pressure of 1 bar was 1.40microns. In addition, the powder displayed flowrate independent behavioras can be seen from the ratio of ×50 measured at 0.5 bar to ×50 measuredat 4.0 bar, which was 1.04. The value for 1/4 bar for these particleswas 1.00, demonstrating the that particles were highly dispersable.

TABLE 25 Summary of ACI-2 data for the three batches of particles forFormulation IV. Formulation: IV-A IV-B IV-C FPF less than 5.6 μm onACI-2 (%) 71.3 66.6 68.2 FPF less than 3.4 μm on ACI-2 (%) 47.5 44.848.7

Additional information relating to properties of the Formulation IVpowder and/or particles prepared in this example are provided in theTables or graphs shown in FIGS. 1A-1F

Example 15 Dispersibility

This example demonstrates the dispersibility of dry powder formulationscomprising calcium lactate, calcium sulfate or calcium citrate powderswhen delivered from different dry powder inhalers over a range ofinhalation maneuvers and relative to a traditional micronized drugproduct similarly dispersed.

The dispersibility of various powder formulations was investigated bymeasuring the geometric particle size and the percentage of powderemitted from capsules when inhaling on dry powder inhalers with flowrates representative of patient use. The particle size distribution andweight change of the filled capsules were measured for multiple powderformulations as a function of flow rate, inhaled volume and fill weightin 2 passive dry powder inhalers.

Powder formulations were filled into size 3 HPMC capsules (CapsugelV-Caps) by hand with the fill weight measured gravimetrically using ananalytical balance (Mettler Tolerdo XS205). Fill weights of 25 and 35 mgwere filled for Formulation I (lot #26-190-F), 25, 60 and 75 mg forFormulation III (Lot#69-191-1), 25 and 40 mg for Formulation II (Lot#65-009-F), 10 mg for a spray dried leucine powder (lot#65-017-F) and 25mg of micronized albuterol sulfate (Cirrus lot#073-001-02-039A). Twocapsule based passive dry powder inhalers (RS-01 Model 7, Low resistancePlastiape S.p.A. and RS-01 Model 7, High resistance Plastiape S.p.A.)were used which had specific resistances of 0.020 and 0.036 kPa1/2/LPMwhich span the typical range of dry powder inhaler resistance. Flow rateand inhaled volume were set using a timer controlled solenoid valve withflow control valve (TPK2000, Copley Scientific). Capsules were placed inthe appropriate dry powder inhaler, punctured and the inhaler sealed tothe inlet of the laser diffraction particle sizer (Spraytec, Malvern).The steady air flow rate through the system was initiated using theTPK2000 and the particle size distribution was measured via the Spraytecat 1 kHz for the durations at least 2 seconds and up to the totalinhalation duration. Particle size distribution parameters calculatedincluded the volume median diameter (Dv50) and the geometric standarddeviation (GSD) and the fine particle fraction (FPF) of particles lessthan 5 micrometers in diameter. At the completion of the inhalationduration, the dry powder inhaler was opened, the capsule removed andre-weighed to calculate the mass of powder that had been emitted fromthe capsule during the inhalation duration. At each testing condition, 5replicate capsules were measured and the results of Dv50, FPF andcapsule emitted powder mass (CEPM) were averaged.

In order to relate the dispersion of powder at different flow rates,volumes, and from inhalers of different resistances, the energy requiredto perform the inhalation maneuver was calculated and the particle sizeand dose emission data plotted against the inhalation energy. Inhalationenergy was calculated as E=R²Q²V where E is the inhalation energy inJoules, R is the inhaler resistance in kPa¹²/LPM, Q is the steady flowrate in L/min and V is the inhaled air volume in L.

FIG. 14 shows the dose emitted from a capsule for Formulation III powderat 3 different capsule fill weights, using both the high resistance andlow resistance RS-01 dry powder inhalers. At each fill weight, steadyinhalations ranged from a maximum energy condition of 9.2 Joules whichwas equivalent to a flow rate of 60 L/min through the high resistanceinhaler (R=0.036 kPa^(1/2)/LPM) with a total volume of 2 L down to lowerenergies with reduced volumes down to 1 L, reduced flow rates down to 15L/min and inhaler resistance down to R=0.020 kPa¹²/LPM. As can be seenfrom FIG. 14, the entire mass of powder filled into the capsule emptiesout of the capsule in a single inhalation for all 3 fill weights of 25,60 and 75 mg of Formulation III at the highest energy condition tested.For the 25 mg fill weight, greater than 80% of the fill weight emptieson average for all inhalation conditions down to 0.16 Joules. At 60 mg,the capsule dose emission drops below 80% of the fill weight at 0.36Joules. At a capsule fill weight of 75 mg, the capsule dose emissiondrops below 80% of the fill weight at 1.2 Joules.

Also shown in FIG. 14 are 2 fill weights of 25 mg and 40 mg of amicronized albuterol sulfate drug formulation which was jet milled to anaverage particle size of 1.8 micrometers, hand filled into size 3capsules and dispersed in the high resistance RS-01 inhaler. As can beseen for both the 25 and 40 mg fill weights, at an inhalation energy of9.2 Joules (steady inhalation of 60 L/min for 2 L) the average CEPM isabove 80% of the capsule fill weight (93% for the 25 mg fill weight and84% for the 40 mg fill weight). However, at all measured lower energies,the CEPM drops to below 10 mg (<30% of capsule fill weight) for bothfill weights and monotonically decreases with decreases in inhalationenergy.

FIG. 15 shows the particle size distribution of the Formulation IIIpowders that are emitted from the inhalers characterized by the volumemedian diameter (Dv50) and plotted against the inhalation energyapplied. Consistent values of Dv50 at decreasing energy values indicatethat the powder is well dispersed since additional energy does notresult in additional deagglomeration of the emitted powder. The Dv50values are consistent for all three fill weights of 75, 60 and 25 mg atall high energy values, with the Dv50 remaining below 2 micrometers downto 0.51 Joules for all 3 fill weights (FIG. 15). Taking into accountthat at the 60 and 75 mg fill weights, inhalations in the 0.5 to 1.2Joule range did not fully emit the powder from the capsule (FIG. 14), itis clear that the powder which was emitted was still fully dispersed bythe DPI (FIG. 15). In this range, the Dv50 is not significantlyincreased in size, which would be expected if the emitting powdercontained a lot of agglomerates and was not well dispersed.

Also shown in the FIG. 15 are fill weights of 25 mg (x) and 40 mg (+) ofa micronized albuterol sulfate drug formulation which was jet milled toan average particle size of 1.8 micrometers, hand filled into size 3capsules and dispersed in the high resistance RS-01 inhaler. As can beseen for both the 25 and 40 mg fill weights, at an inhalation energy of9.2 Joules (steady inhalation of 60 L/min for 2 L) the average Dv50 isbelow 2 micrometers (1.8 and 1.6 μm respectively) for both fill weights,demonstrating good dispersion and relatively few agglomerates. However,at all measured lower energies, the Dv50 increases to greater than 2micrometers (3.9 and 3.1 μm respectively) and continues to monotonicallyincrease with decreasing inhalation energy, demonstrating agglomerationand poor dispersion of the primary particles.

Additional powders were tested at all of the test conditions and averageCEPM and Dv50 were determined (Table 26) These results demonstrate theability of the powder formulations to be fully emptied anddeagglomerated at inhalation energies down to approximately 0.5 Joules.

TABLE 26 Mean CEPM, Dv(50) and FPF as a function of fill weight,flowrate and duration for Formulations I to III, placebo, and micronizedalbuterol sulfate. Inhalation Mean Fill Flow Energy, Mean Mean FPF,Weight Rate Duraton E = R²Q²V CEPM Dv(50) % Powder DPI (mg) (LPM) (s)(Joules) (mg) (μm) <5 μm Formulation RS.01.HR 25 15 4 0.29 15.84 4.7752.09 I Formulation RS.01.HR 25 20 3 0.51 22.88 3.46 65.79 I FormulationRS.01.HR 25 30 2 1.15 24.75 2.94 72.88 I Formulation RS.01.HR 25 60 29.18 24.72 2.93 73.39 I Formulation RS.01.LR 25 15 4 0.09 4.30 7.2931.97 I Formulation RS.01.LR 25 20 3 0.16 8.05 5.10 48.98 I FormulationRS.01.LR 25 30 2 0.36 19.94 3.28 71.09 I Formulation RS.01.LR 25 60 22.85 24.75 2.51 80.26 I Formulation RS.01.HR 35 30 2 1.15 33.77 2.1783.17 I Formulation RS.01.HR 35 60 2 9.18 34.73 2.33 81.42 I FormulationRS.01.LR 35 30 2 0.36 13.07 3.16 73.22 I Formulation RS.01.LR 35 60 22.85 34.57 2.34 83.15 I Placebo RS.01.HR 10 15 4 0.29 3.87 25.71 6.22Placebo RS.01.HR 10 20 3 0.51 8.79 22.80 8.64 Placebo RS.01.HR 10 30 21.15 9.42 22.95 11.83 Placebo RS.01.HR 10 60 2 9.18 9.78 21.45 12.52Placebo RS.01.LR 10 15 4 0.09 1.87 40.36 3.17 Placebo RS.01.LR 10 20 30.16 3.08 28.16 5.20 Placebo RS.01.LR 10 30 2 0.36 7.01 18.62 9.39Placebo RS.01.LR 10 60 2 2.85 9.82 15.26 16.41 Formulation RS.01.HR 2515 4 0.29 24.87 3.26 68.77 III Formulation RS.01.HR 25 20 3 0.51 25.483.06 72.61 III Formulation RS.01.HR 25 30 2 1.15 25.05 2.90 74.06 IIIFormulation RS.01.HR 25 60 2 9.18 25.28 2.92 71.87 III FormulationRS.01.LR 25 15 4 0.09 18.97 5.59 43.81 III Formulation RS.01.LR 25 20 30.16 24.95 3.45 68.14 III Formulation RS.01.LR 25 30 2 0.36 25.08 2.7276.82 III Formulation RS.01.LR 25 60 2 2.85 24.88 2.66 75.76 IIIFormulation RS.01.HR 40 30 2 1.15 39.55 2.76 74.92 III FormulationRS.01.HR 40 60 2 9.18 40.13 3.14 67.35 III Formulation RS.01.LR 40 30 20.36 39.74 2.89 75.51 III Formulation RS.01.LR 40 60 2 2.85 39.85 2.6577.00 III Formulation RS.01.HR 25 15 4 0.29 24.45 3.56 63.96 IIFormulation RS.01.HR 25 17.5 3.4 0.39 21.43 2.34 80.07 II FormulationRS.01.HR 25 20 3 0.51 23.55 2.15 82.08 II Formulation RS.01.HR 25 25 2.40.80 24.42 1.39 90.70 II Formulation RS.01.HR 25 30 2 1.15 24.88 1.2888.29 II Formulation RS.01.HR 25 60 2 9.18 25.07 1.59 85.28 IIFormulation RS.01.LR 25 15 4 0.09 7.47 7.46 32.20 II FormulationRS.01.LR 25 20 3 0.16 20.39 4.29 57.09 II Formulation RS.01.LR 25 30 20.36 24.23 2.52 78.85 II Formulation RS.01.LR 25 60 2 2.85 24.81 1.6189.78 II Formulation RS.01.HR 60 25 2.4 0.80 52.42 0.99 90.45 IIFormulation RS.01.HR 60 30 2 1.15 56.50 0.78 92.70 II FormulationRS.01.HR 60 60 2 9.18 59.42 1.19 90.64 II Formulation RS.01.LR 60 30 20.36 26.62 2.48 80.08 II Formulation RS.01.LR 60 60 2 2.85 59.51 1.1990.64 II Formulation RS.01.HR 75 25 2.4 0.80 47.63 1.36 89.83 IIFormulation RS.01.HR 75 30 2 1.15 51.84 1.07 92.59 II FormulationRS.01.HR 75 60 2 9.18 74.90 1.41 85.20 II Micronized RS.01.HR 25 15 40.29 3.12 16.76 13.00 Albuterol Micronized RS.01.HR 25 20 3 0.51 5.008.40 32.10 Albuterol Micronized RS.01.HR 25 30 2 1.15 7.08 3.86 59.44Albuterol Micronized RS.01.LR 25 60 2 2.85 15.28 2.57 75.01 AlbuterolMicronized RS.01.HR 25 60 2 9.18 23.18 1.77 81.65 Albuterol MicronizedRS.01.HR 40 15 4 0.29 2.43 17.63 10.73 Albuterol Micronized RS.01.HR 4020 3 0.51 4.97 6.34 42.24 Albuterol Micronized RS.01.HR 40 30 2 1.158.55 3.13 67.18 Albuterol Micronized RS.01.LR 40 60 2 2.85 18.88 2.6273.98 Albuterol Micronized RS.01.HR 40 60 2 9.18 33.40 1.60 84.30Albuterol

Example 16 Solid State Particle Analysis

A. X-Ray Powder Diffraction

Formulations I, II, III and IV were analyzed for amorphous/crystallinecontent and polymorphic form using high resolution X-ray powderdiffraction (XRPD) and differential scanning calorimetry (DSC). ForXRPD, phase identification was performed to identify any crystallinephases observed in each XRPD pattern. XRPD patterns were collected usinga PANalytical X'Pert Pro diffractometer (Almelo, The Netherlands). Thespecimen was analyzed using Cu radiation produced using an Optix longfine-focus source. An elliptically graded multilayer mirror was used tofocus the Cu Kα X-rays of the source through the specimen and onto thedetector. The specimen was sandwiched between 3-micron thick films,analyzed in transmission geometry, and rotated to optimize orientationstatistics. A beam-stop was used, along with helium purge in some cases,to minimize the background generated by air scattering. Soller slitswere used for the incident and diffracted beams to minimize axialdivergence. Diffraction patterns were collected using a scanningposition-sensitive detector (X'Celerator) located 240 mm from thespecimen. The data-acquisition parameters of each diffraction patternare displayed above the image of each pattern in appendix C. Prior tothe analysis a silicon specimen (NIST standard reference material 640c)was analyzed to verify the position of the silicon 111 peak. Calculatedpatterns for the potential crystalline components (including anhydrousand hydrated forms) were produced from either the Cambridge StructuralDatabase or the International Center for Diffraction Data (ICDD)Database and compared to the experimental patterns. The crystallinecomponents were qualitatively determined. XRPD was also performed onpowders that had been conditioned at 75% RH for a period of three tofour hours in a Dynamic Vapor Sorption system in order to assess thepropensity for recrystallization of said powders upon short-termexposure to elevated humidities.

Differential scanning calorimetry (DSC) was performed using a TAInstruments differential scanning calorimeter Q2000 (New Castle, Del.).The sample was placed into an aluminum DSC pan, and the weightaccurately recorded. The data acquisition and processing parameters aredisplayed on each thermogram. Indium metal was used as the calibrationstandard. The glass transition temperature (T_(g)) is reported from theinflection point of the transition/or/the half-height of the transition.Standard mode DSC experiments were initially conducted on the powders ofinterest in order to assess the overall thermal behavior of the powders.Cyclic mode DSC experiments were also performed in order to attempt toidentify the occurrence of glass transitions occurring in these powdersover temperature regions of interest identified in the standard DSCthermograms.

Surprisingly, high calcium and sodium salt content powders were producedthat possessed a mixture of amorphous and crystalline content thatpossessed optimized properties with respect to their dispersibility andstability in the dry state and their dissolution and water absorptionproperties in the hydrated state. As shown in FIGS. 16 and 17, theFormulation I powder was observed via XRPD to consist of a combinationof crystalline sodium chloride and a poorly crystalline or amorphouscalcium citrate and potentially calcium chloride-rich phase (asevidenced by a lack of observance of any characteristic peaks for anycalcium salt forms in this powder as well as the absence of anycharacteristic peaks for leucine). As shown in FIG. 18, a glasstransition temperature of approximately 167° C. was observed via cyclicDSC for the amorphous calcium-rich phase, indicating that this amorphousphase should be relatively stable to crystalline conversion at standardconditions (25° C., 30% RH). The presence of crystalline sodium chloridein this powder in the dry state may enhance the dispersibility andstability of said powder. The presence of the calcium salt in a poorlycrystalline or amorphous form in the Formulation I powder may alsofacilitate the rapid water uptake and dissolution properties of theFormulation I formulation upon deposition in the lungs (i.e.,crystalline sodium chloride is readily soluble, whereas calcium citrateis poorly soluble). When a particle or powder is readily soluble, itdissolves rapidly. When a particle or powder is poorly soluble, itdissolves slowly.

Similar results were seen for powders Formulation III and FormulationIV. As shown in FIGS. 19 and 20, the Formulation III powder was observedvia XRPD to consist of a combination of crystalline sodium chloride anda poorly crystalline or amorphous calcium lactate and potentiallycalcium chloride-rich phase (as evidenced by a lack of observance of anycharacteristic peaks for any calcium salt forms in this powder as wellas the absence of any characteristic peaks for leucine). As shown inFIG. 21, a glass transition temperature of approximately 144° C. wasobserved via cyclic DSC for the amorphous calcium-rich phase, indicatingthat this amorphous phase should be relatively stable to crystallineconversion at standard conditions (25° C., 30% RH). Nearly identicalresults were seen for the Formulation IV powder which contained 10%maltodextrin versus 10% leucine (see FIGS. 22 and 23) for XRPD data aswell as FIG. 24 which shows a glass transition temperature ofapproximately 134° C.

In contrast, the Formulation II formulation displayed the presence ofsome degree of crystalline calcium salt content (calcium sulfate) inaddition to crystalline sodium chloride (see FIGS. 25A and 25B).However, this powder still possessed a significant degree of amorphous,calcium-rich phase content, as evidenced by the presence of a glasstransition temperature of approximately 159° C. via DSC (see FIG. 26).

B. Surface RAMAN Mapping

Surface Mapping RAMAN experiments were conducted on samples ofFormulations I through IV in order to determine the nature of thechemical composition at the surface of the particles comprising theseformulations. Raman map spectra were acquired on a Renishaw inViaRamascope (Gloucestershire, UK) equipped with a Leica DM LM microscope(Wetzlar, Germany). The instrument was calibrated using a silicon waferstandard. The samples were prepared for analysis on an aluminum-coatedmicroscope slide. The excitation wavelength was 785 nm using ahigh-power near-infrared diode laser source. The data collection forFormulation I, Formulation III and Formulation IV was a static scan witha 30 second exposure time and 10 accumulations. The data collection forFormulation II was an extended scan with a 60 second exposure time andone accumulation. A Philips ToUcam Pro II camera (model PCVC 840K)(Amsterdam, the Netherlands) was used for image acquisition with a 50×objective. Renishaw WiRE 3.1 (service pack 9) software (Gloucestershire,UK) was used for data collection and processing.

Raman spectra were acquired for six particles from the Formulation Isample, and are shown overlaid in FIG. 27A. Spectra files 389575-1 and389575-6 are characterized by the presence of weak peaks atapproximately 1450, 965 and 850 cm−1. These peaks are discernable asonly very weak features in spectra file 389575-6, and are not detectedin the remaining spectral data files. In FIG. 27B, spectrum 389575-6 isbackground subtracted and overlaid with the Raman spectra of calciumcitrate tetrahydrate, sodium citrate, and leucine. The sample spectrumexhibits peaks at approximately 1450 and 850 cm−1 which are common toboth leucine and the citrate salts. The sample spectrum displays anadditional peak at approximately 965 cm−1, which is consistent with therelatively stronger intensity peak in the spectrum of the citrate salts(i.e., calcium citrate tetrahydrate and sodium citrate). Thecharacteristic leucine peak at 1340 cm−1 is not observed in the samplespectra.

Raman spectra were acquired for eight particles from the Formulation IIsample, and are shown overlaid in FIG. 27C. All particle spectra arecharacterized by the presence of a peak at approximately 1060 cm−1. Anadditional peak at approximately 670 cm−1 is observed in spectral file388369-4. The 670 cm−1 peak is also observable in spectral data files388369-1, 3, and 8 after background subtraction (not shown). In FIG.27D, spectrum 388369-4 is background subtracted and overlaid with theRaman spectra of calcium sulfate, calcium sulfate dihydrate, sodiumsulfate anhydrous, and leucine. The background subtracted samplespectrum reveals a possible third peak near 520 cm−1. The peaks at 1060and 670 cm−1 are present at similar positions to characteristic peaks ofthe sulfate ions displayed, but do not overlap precisely. Thefrequencies of the peaks at 1060 and 670 cm−1 in the sample spectrum areconsistent with the stretching and bending modes, respectively, of asulfate ion functional group. Peaks assignable to leucine are notdetected in the particle spectra.

Raman spectra were acquired for twelve particles from the FormulationIII sample, and are shown overlaid in FIG. 27E. All particle spectra arecharacterized by the presence of peaks at approximately 1045 and 860cm−1. Additional peaks can be observed in various spectra atapproximately 1450, 1435, 1125, 1095, 930, and 775 cm−1, which generallycorrelate in relatively intensity with the strong peak at 1045 cm−1. InFIG. 27F, spectra 389576-7 and 389576-12 are background subtracted andoverlaid with the Raman spectra of calcium lactate pentahydrate, andleucine. A good correspondence is observed between the sample spectraand calcium lactate pentahydrate spectrum. However, the sample spectradisplay additional weak peaks at approximately 1345, 1170, 960, 830, and760 cm−1 which are absent in the spectrum of calcium lactatepentahydrate. Similar peaks are present in the reference spectrum ofleucine, although with slightly different relative intensities andfrequencies.

Raman spectra were acquired for twelve particles from the Formulation IVsample, and are shown overlaid in FIG. 27G. All particle spectra arecharacterized by the presence of a peak at approximately 1045 cm−1. Allparticle spectra except file 389577-2 also display a peak atapproximately 860 cm−1. Additional peaks can be observed in variousspectra at approximately 1450, 1435, 1125, 1095, 930, and 775 cm−1,which generally correlate in relatively intensity with the strong peakat 1045 cm−1. In FIG. 27H, spectrum 389577-9 is background subtractedand overlaid with the Raman spectra of calcium lactate pentahydrate. Agood correspondence is observed between the sample and calcium lactatepentahydrate spectra. Peaks assigned to maltodextrin (not shown) are notobserved in the sample spectra.

Thus, RAMAN surface mapping analysis indicates that the surfacecomposition of each of Formulations I though IV is dominated by thepresence of the various calcium salts (calcium citrate for FormulationI, calcium sulfate for Formulation II and calcium lactate forFormulations III and IV). For the case of Formulations I through III,this is in contrast to the reported use of leucine as adispersion-enhancing agent that increases the dispersibility of powdersfor aerosolization via being concentrated at the surface of theparticles comprising said powders. For the formulations disclosedherein, it does not appear that leucine is acting as a dispersibilityenhancer in this fashion, as also evidenced by the similar results seenfor Formulations III (leucine-containing calcium lactate formulation)and IV (maltodextrin-containing calcium lactate formulation) withrespect to surface content and dispersibility.

Example 17 Ion Exchange Reaction for Spray Drying Supersaturated CalciumCitrate and Calcium Sulfate

Saturated or super-saturated stocks of aqueous calcium sulfate orcalcium citrate were prepared for spray drying using calcium chlorideand sodium sulfate or calcium chloride or sodium citrate as startingmaterials. A range of total solids concentrations from 5 to 30 g/L wereprepared both by (i) pre-mixing both salts in water and (ii) keeping thecalcium and sodium salt in separate aqueous solutions, with staticmixing in-line immediately before spray drying. All of the liquid feedstocks prepared contained saturated or supersaturated calcium sulfateamounts, (where the solubility limit of calcium sulfate in water is 2.98g/L) and saturated or supersaturated calcium citrate amounts (where thesolubility limit of calcium citrate in water is 0.96 g/L). Consideringthe calcium chloride and sodium sulfate precipitation reaction proceedsto completion (CaCl₂+Na₂SO₄→CaSO₄+2NaCl), the corresponding finalconcentrations of calcium sulfate are listed in Table 24. Similarresults for the calcium chloride and sodium citrate precipitationreaction (3CaCl₂+2 Na₃C₆H₅O₇→Ca₃(C₆H₅O₇)₂+6 NaCl) are also shown inTable 27.

TABLE 27 Liquid feedstock total solids concentrations and final calciumsulfate or calcium citrate concentrations, where the aqueous solubilitylimit of calcium sulfate is 2.98 g/L and calcium citrate is 0.96 g/LTotal solids Final calcium sulfate Final calcium citrate concentrationconcentration concentration (g/L) (g/L) (g/L) 5 2.7 2.9 10 5.4 5.9 158.1 8.8 20 10.8 11.7 30 16.1 17.6

Formulations of 44 weight percent calcium chloride and 56 weight percentsodium sulfate were produced by spray drying utilizing a Mobile Minorspray dryer (Niro, GEA Process Engineering Inc., Columbia, Md.). Theliquid feed stocks were prepared at a range of solids concentration from5-30 g/L. For pre-mixed feeds, sodium salt then calcium salt wasdissolved in DI water with constant stirring on a magnetic stirplate.For static mixed feeds, calcium salt was dissolved in DI water, andsodium salt was separately dissolved in DI water with the two solutionsmaintained in separate vessels with constant agitation. Atomization ofthe liquid feed was performed using a co-current two-fluid nozzle (Niro,GEA Process Engineering Inc., Columbia, Md.). The liquid feed was fedusing gear pumps (Cole-Parmer Instrument Company, Vernon Hills, Ill.)either directly into the two-fluid nozzle for pre-mixed feeds or into astatic mixer (Charles Ross & Son Company, Hauppauge, N.Y.) immediatelybefore introduction into the two-fluid nozzle for static mixed feeds.Nitrogen was used as the drying gas and dry compressed air as theatomization gas feed to the two-fluid nozzle. The process gas inlettemperature was 240-250° C. and outlet temperature was 94-98° C. with aliquid feedstock rate of 50-70 mL/min. The gas supplying the two-fluidatomizer was approximately 11 kg/hr. The pressure inside the dryingchamber was at −2 “WC. Spray dried product was collected from a cycloneand analyzed for volume particle size by laser diffraction using a HELOSwith RODOS attachment and for aerosol properties using a collapsedtwo-stage ACI.

Pre-mixed feeds were assessed for solution stability and clarity. At atotal solids concentration of 5 g/L, where the final calcium sulfateconcentration would be slightly over the solubility limit of calciumsulfate, the solution stayed clear during the 30 minute duration ofmixing and spray drying. As the total solids concentration increased andthe final calcium sulfate concentration greatly exceeded the solubilitylimit, the feed stock became cloudy and precipitation was evident. At 10g/L the liquid was slightly cloudy, at 20 g/L the liquid was clear forapproximately 5-10 minutes before becoming increasingly cloudy over thecourse of 10 minutes and at 30 g/L the liquid was clear forapproximately 2 minutes after mixing, with visible precipitationappearing after approximately 5 minutes.

The pre-mixed and static mixed liquid feed stocks were spray dried andthe resulting dry powder collected from the cyclone. Results from theHELOS with RODOS are shown in FIG. 28 with representative particle sizedistributions shown in FIG. 29. While an increase in particle size isexpected with increasing feed stock solids concentrations (as seen inthe static mixed feeds), the significant particle size increase andbroadened particle size distribution in the pre-mixed feeds isundesirable.

Results for aerosol characterization of the dry powders using thecollapsed ACI are shown in FIG. 30.

Unstable solutions with continued precipitation may negatively affectreproducible particle formation during spray drying and also result in abroad particle size distribution. The supersaturated, clear solutionsevident for 2-10 minutes for the higher solids concentration suggestthat the solutions could be static mixed to achieve a higher spraydrying throughput while reproducibly producing a narrow particle sizedistribution.

Similar results were exemplified for calcium citrate, as demonstrated inExample 1 for the formulation comprising 10.0 weight percent leucine,35.1 weight percent calcium chloride and 54.9 weight percent sodiumcitrate (Formulation I-A). The precipitation reaction will result in aformulation comprising 10.0 weight percent leucine, 52.8 weight percentcalcium citrate and 37.2 weight percent sodium chloride. At a totalsolids concentration of 10 g/L, the final calcium citrate concentrationwould be 5.3 g/L, which exceeds the solubility limit of calcium citratein water of 0.96 g/L. As can be seen from the properties of the spraydried powder (FIGS. 1A-1E and 2-4), this supersaturated solutionresulted in respirable particles with narrow size distribution.

Example 18

Small, dispersible particles were made from calcium-containingformulations with and without leucine, as well as magnesium-containingand sodium only formulations.

The following powders were spray dried on the Büchi B-290 using the highperformance cyclone with an air feed rate of 30 mm air, aspirator at 90%rate and the small glass collection vessel. The inlet temperature was220° C. and the outlet temperature was between 96-102° C. The solidsconcentration was 5 g/L and all were mixed in D.I. water by fullydissolving one component at a time, before adding the next in the orderin which they are listed.

18-1) 10.0% lactose, 30.6% magnesium chloride, 59.4% sodium citrate,Ca:Na ratio=1:2

18-2) 63.4% magnesium lactate, 36.6% sodium chloride, Ca:Na ratio=1:2

18-3) 10.0% leucine, 58.4% magnesium lactate, 31.6% sodium chloride,Ca:Na ratio=1:2

18-4) 50.0% leucine, 50% calcium lactate

18-5) 10% leucine, 90% sodium chloride

18-6) 60% leucine, 40% sodium chloride

18-7) 10.0% albuterol, 58.6% calcium lactate, 31.4% sodium chloride

18-8) 90.0% albuterol, 5.9% calcium lactate, 3.1% sodium chloride

Characterization results for these powders are shown in Table 28 below.All eight powders exhibited good dispensability with respect to ×500.5/4 and 1/4 ratios. FPF's<5.6 microns ranged from a low of 18.7% to75.6%.

TABLE 28 Assorted sodium, calcium and magnesium-based formulations. x50(μm) GSD FPF_TD FPF_TD @ @ 1/4 0.5/4 <3.4 um <5.6 um % Mass yieldFormulation Method 1 bar 1 bar bar bar % % collected % lact: Buchi 2.92.3 1.1 1.1 18.1% 37.8% 55.7% 88.9% MgCl2: HP Na3Cit 10:30.6:59.4leucine: Buchi 2.7 2.4 0.8 1.1 14.5% 32.3% 53.0% 80.0% MgLact: HP NaCl10:58.6:31.4 MgLact: Buchi 3.3 2.1 1.0 1.0 16.5% 39.3% 59.8% 78.0% NaClHP 63.4:36.6 leu:CaLact Buchi 3.5 2.2 1.1 1.1 19.2% 38.5% 60.4% 76.0%50:50 HP leu:NaCl Buchi 1.1 1.7 1.0 1.2 53.0% 71.0% 78.6% 67.9% 10:90 HPleu:NaCl Buchi 1.4 2.2 1.1 1.2 49.7% 75.6% 85.2% 54.3% 60:40 HPalbuterol: Buchi 2.8 2.3 0.9 1.0 16.0% 38.6% 60.2% 81.5% CaLact:NaCl HP10:58.6:31.4 albuterol: Buchi 3.5 2.3 1.0 1.1  8.9% 18.7% 29.1% 40.5%CaLact:NaCl HP 90:5.9:3.1

Several additional calcium-free exemplary formulations were producedutilizing various spray-dryer systems (Buchi, LabPlant and Niro systems)following similar procedures those described above. Selectedcharacterization results for the resultant powders are shown in Table 29(cells with blank values indicates no value was measured for thatpowder).

TABLE 29 Non-calcium formulations of small, dispersible powders x50 (μm)GSD FPF_TD FPF_TD @ 1 @ 1 1/4 0.5/4 water <3.4 um <5.6 um % Mass yieldLot Formulation Method bar bar bar bar % % % collected % NaCl 2.26.2NaCl, 100 Labplant 2.9 1.4 0.5% 27.115.4 NaCl 100 Niro 4.5 1.9 1.4 0.6%5.2% 22.0% 43.1% 61.3% Magnesium Salts 27.33.2 MgCl2 + NaCl Labplant 4.31.9 1.2 29.9% 2.3% 5.7% 14.0% 17.9% 27.15.4 MgCl2:Na2CO3, 47:53 Labplant2.3 1.4 1.1 87.4% 17.6% 68.124.1 lactose:MgCl2:Na3Cit Buchi 2.9 2.3 1.11.1 18.1% 37.8% 55.7% 88.9% 10:30.6:59.4 HP 68.129.1 leucine:MgLact:NaClBuchi 2.7 2.4 0.8 1.1 14.5% 32.3% 53.0% 80.0% 10:58.6:31.4 HP 68.129.2MgLact:NaCl Buchi 3.3 2.1 1.0 1.0 16.5% 39.3% 59.8% 78.0% 63.4:36.6 HPLeucine 26.155.1 Leucine, 100 Buchi 4.1 2.3 1.1 33.6% 58.5% 71.8% 56.7%HP

Further, several additional examples of compositions containing eitherno excipients or non-leucine excipients were also produced utilizingvarious spray-dryer systems (Buchi, Labplant and Niro-based systems)following similar procedures those described above. Selectedcharacterization results for the resultant powders are shown in Table 30(cells with blank values indicates no value was measured for thatpowder).

TABLE 30 Non-leucine salt formulations of small, dispersible powders x50(μm) GSD FPF_TD FPF_TD @ 1 @ 1 1/4 0.5/4 water <3.4 um <5.6 um % Massyield Lot Formulation Method bar bar bar bar % % % collected %Excipients with lactate 45.132.1 leu:mdextrin:CaLact:NaCl Buchi 1.5 1.91.0 1.0 31.8% 53.7% 62.9% 65.6% 5:5:58.6:31.4 HP 45.137.1lact:CaLact:NaCl Buchi 2.7 2.0 1.0 1.0 8% 24.9% 48.1% 63.4% 81.4%10:58.6:31.4 HP 45.137.2 mannitol:CaLact:NaCl Buchi 1.5 6% 43.6% 66.6%73.1% 68.6% 10:58.6:31.4 HP 45.189.2 mannitol:CaLact:NaCl Buchi 1.2 1.81.0 1.0 5% 44.8% 66.0% 71.6% 10:58.6:31.4 HP 45.137.3mdextrin:CaLact:NaCl Buchi 1.4 1.9 1.0 1.0 6% 47.5% 71.3% 77.6% 77.7%10:58.6:31.4 HP 45.189.3 mdextrin:CaLact:NaCl Buchi 1.3 1.8 1.0 1.0 7%44.8% 66.6% 73.2% 10:58.6:31.4 HP 45.137.4 trehalose:CaLact:NaCl Buchi1.4 1.9 1.0 1.0 4% 51.3% 72.8% 78.2% 77.2% 10:58.6:31.4 HP CalciumCitrate 2.26.3 CaCl2:Na3Cit 39:61 Labplant 3.3 1.2 1.0 11.0% 22.8%26.048.2 CaCl2:Na3Cit2 39:61 Niro 7.0 2.1 1.2 7.9% 22.0% 46.1% 61.0%27.03.1 CaCl2:Na3Cit 39:61 Labplant 3.6 1.4 1.1 9.0% 25.1% 26.013.3CaCl2:Na3Cit 49:51 Niro 3.6 2.0 1.1 12.7% 31.0% 45.9% 43.9% not tocompletion 27.183.4 Ca(OH)2:Cit acid:NaCl Buchi 2.6 1.8 1.0 9.3% 17.7%21.5% 23.1% 35:61:3.5 Calcium Sulfate 2.26.4 CaCl2:Na2SO4 44:56 Labplant3.7 1.7 1.4 5.1% 12.1% 26.060.1 CaCl2:Na2SO4 44:56 Niro 3.0 2.0 1.315.3% 40.2% 62.9% 60.8% 26.060.3 CaCl2:Na2SO4 44:56- Niro 2.6 1.6 1.217.0% 42.5% 58.6% 31.4% static mixed 26.069.1 CaCl2:NaSO2 44:56 5 g/LNiro 2.9 1.6 1.4 11.1% 38.5% 59.1% 25.2% 26.069.2 CaCl2:NaSO2 44:56 10g/L Niro 3.5 1.8 1.5 7.6% 27.7% 61.1% 45.6% 26.069.3 CaCl2:NaSO2 44:5620 g/L Niro 4.0 2.1 1.4 6.9% 25.3% 62.6% 37.3% 26.124.1 CaCl2:Na2SO4,44:56 5 g/L Niro 2.9 1.5 1.5 6.5% 11.0% 34.5% 53.4% 22.0% 26.124.2CaCl2:Na2SO4, 44:56 10 g/L Niro 3.2 1.5 1.7 7.1% 9.9% 28.9% 45.1% 35.0%27.114.5 CaCl2:Na2SO4 44:56 Niro 4.1 1.8 1.6 6.8% 5.8% 22.6% 50.2% 52.5%27.154.1 CaCl2:Na2SO4 44:56 Buchi 3.1 1.9 1.3 14.0% 31.6% 55.1% 50.3%27.114.6 CaCl2:Na2SO4:Rhod B Niro 3.9 1.9 1.0 7.2% 7.4% 25.5% 52.4%44.2% 44:56.1 27.114.1 lact:CaCl2:Na2SO4 Niro 3.9 2.5 1.2 17.9% 12.0%28.5% 42.5% 13.3% 90:4.4:5.6 27.114.2 lact:CaCl2:Na2SO4 Niro 4.5 2.0 1.112.6% 10.2% 29.1% 44.5% 58.0% 50:22:28 27.115.3 CaSO4 100 Niro 3.8 1.71.2 14.0% 15.8% 38.2% 57.0% 47.5% 27.185.2 Ca(OH)2:Sulf acid:NaCl Buchi2.5 1.8 1.3 17.5% 45.2% 65.2% 44.1% 41.3:54.6:4.1 27.185.3 Ca(OH)2:Sulfacid 43:57 Buchi 2.9 2.3 1.1 15.3% 38.9% 59.4% 16.1% 27.183.1CaLact:NaCl 96.8:3.2 Buchi 3.1 2.0 1.1 22.4% 50.9% 69.5% 35.0% 27.115.2CaCl2:Na2CO3 51:49 Niro 3.9 2.1 1.4 1.7% 8.4% 22.4% 38.9% 27.3% 27.184.3CaGluc:NaCl 98.3:1.7 Buchi 2.9 2.0 1.0 13.5% 26.7% 48.3% 47.6% 27.15.2MgCl2:Na3Cit, 36:64 Labplant 3.1 1.4 1.0 13.2% 28.6% 27.33.3MgCl2:Na3Cit, 36:64 Labplant 4.0 2.2 1.2 15.7% 21.4% 53.7% 68.2% 26.2%27.15.3 MgCl2:Na2SO4, 40:60 Labplant 3.9 2.3 1.3 11.1% 31.8% 27.33.9MgCl2:Na2CO3, 47:53 Labplant 2.7 3.7 1.4 7.9% 21.0% 46.0% 58.3% 18.8%27.15.4 MgCl2:Na2CO3, 47:53 Labplant 2.3 1.4 1.1 87.4% 17.6% 68.124.1lact:MgCl2:Na3Cit Buchi 18.1% 37.8% 55.7% 88.9% 10:30.6:59.4 HP 68.129.2MgLact:NaCl 63.4:36.6 Buchi 16.5% 39.3% 59.8% 78.0% HP

Table 31 contains characterization data for additional leucine andcalcium containing small and dispersible powder compositions made viausing a Buchi or a Niro spray-drying system per procedures similar tothose described above (cells with blank values indicates no value wasmeasured for that powder).

TABLE 31 Leucine and calcium-containing formulations of small,dispersible particles x50 (μm) GSD FPF_TD FPF_TD Tapped @ 1 @ 1 1/40.5/4 water <3.4 um <5.6 um % Mass density Lot Formulation Method barbar bar bar % % % collected yield % (g/cc) Chloride 26.010.2leu:CaCl2:NaCl Niro 4.8 2.2 1.1 15.8% 35.9% 50.8% 64.1% 50:29.5:20.526.041.3 leu:CaCl2:NaCl Niro 4.9 2.4 14.7% 28.0% 43.0% 50.2%50:29.5:20.5 Citrate 26.013.1 leu:CaCl2:Na3Cit2 Niro 4.2 2.1 1.6 16.8%35.2% 53.8% 56.1% 50:19.5:30.5 26.013.2 leu:CaCl2:Na3Cit2 Niro 4.8 1.81.3 20.8% 39.6% 52.2% 57.5% 10:35.1:54.9 26-190-F Leucine:CaCl2: Niro2.6 1.9 1.2 1.2 45.7% 61.6% 66.3% 74.8% 0.29 Na3Cit2 10.0:35.1: 54.9Sulfate 26.013.4 leu:CaCl2:Na2SO4 Niro 3.7 2.0 1.4 19.6% 39.4% 60.9%73.1% 10:39.6:50.4 26.060.2 leu:CaCl2:Na2SO4 Niro 2.9 1.9 1.2 16.2%35.2% 53.2% 46.5% 0.18 10:39.6:50.4 26.060.4 leu:CaCl2:Na2SO4 Niro 2.91.7 1.3 18.8% 45.1% 64.4% 49.9% 0.17 10:39.6:50.4 27.154.2leu:CaCl2:Na2SO4 Buchi 3.8 1.9 1.1 17.2% 37.5% 55.5% 56.1% 0.3010:39.6:50.4 65-009-F Leucine:CaCl2: Niro 2.5 2.2 1.4 1.5 60.1% 82.7%88.6% 74.2% 0.34 Na2SO4 10.0:39.6: 50.4 26.053.1 leucine:CaCl2: Niro 4.22.0 1.5 3.3% 23.0% 39.6% 52.0% 59.6% Na2SO4 50:22:28 27.114.4leu:CaCl2:Na2SO4 Niro 4.7 1.8 1.9 3.8% 21.2% 44.6% 59.6% 59.6% 50:22:2827.155.1 leu:CaCl2:Na2SO4 Buchi 3.7 1.9 1.2 15.7% 42.9% 68.8% 47.6% 0.3550:22:28 Calcium sulfate 26.019.4 leu:CaSO4 50:50 Niro 4.1 2.1 1.4 11.9%28.0% 56.0% 101.8% Carbonate 26.019.1 leu:CaCl2:NaCO3 Niro 3.4 1.9 1.79.6% 22.2% 35.9% 46.3% 50:25.5:24.5 26.019.2 leu:CaCl2:NaCO3 Niro 2.71.8 1.4 10.6% 23.8% 37.5% 51.0% 10:45.9:44.1 Lactate 26.041.4leu:CaLact:NaCl Niro 5.0 1.9 9.7% 25.9% 46.6% 56.5% 50:36.8:13.127.183.2 Leu:CaLact:NaCl Buchi 3.7 1.8 1.1 24.9% 48.9% 62.7% 34.1%50:48.4:1.6 27.185.1 Leu:CaLact:NaCl Buchi 3.0 1.9 1.0 26.1% 53.7% 70.0%44.8% 10:66.6:23.4 45.19.1 leu:CaLact:NaCl Buchi 3.4 2.3 0.9 5.2% 12.8%29.1% 50.3% 75.6% 0.74 10:66.6:23.4 HP 45.76.1 leu:CaLact:NaCl Buchi 3.82.1 1.0 5.0% 8.6% 20.9% 36.6% 78.5% 10:58.6:31.4 HP 45.78.1leu:CaLact:NaCl Buchi 1.5 1.9 1.1 4.8% 30.6% 53.4% 62.9% 60.8%10:58.6:31.4 HP 45.80.1 leu:CaLact:NaCl Buchi 1.5 1.9 1.1 4.4% 30.3%53.5% 63.8% 71.0% 10:58.6:31.4 HP 45.81.1 leu:CaLact:NaCl Buchi 2.4 2.81.3 7.2% 19.3% 34.1% 44.3% 64.6% 10:58.6:31.4 HP 68.70.1 leu:CaLact:NaClBuchi 1.5 1.9 1.0 42.8% 63.2% 67.8% 73.9% 10:58.6:31.4 HP 65-003-FLeucine:CaLact: Niro 1.5 2.5 1.1 1.1 43.4% 63.5% 69.7% 62.9% 0.69 NaCl10.0: 58.6:31.4 Gluconate 27.184.1 Leu:CaGluc:NaCl Buchi 3.4 2.1 1.035.0% 61.4% 76.3% 51.9% 50:49.15:0.85 27.184.4 leu:CaGluc:NaCl Buchi 3.52.0 1.2 34.1% 60.7% 71.5% 46.3% 50:42.35:7.65 27.184.2 Leu:CaGluc:NaClBuchi 2.7 2.0 1.0 24.9% 52.2% 64.2% 51.0% 10:88.5:1.5

Example 19

Pure calcium chloride was spray dried in the LabPlant spray dryingsystem with an inlet temperature of 180° C. The liquid feed consisted of20 g/L solids concentration of calcium chloride dihydrate in D.I. water.Water condensed in the collection vessel as the calcium chloridedeliquesced and no powder could be collected. Pure calcium chloride wasdeemed too hygroscopic for spray drying from an aqueous solution withhigh water content in the exhaust drying gas. The liquid feed was thenchanged to 70% ethanol to reduce humidity in the exhaust gas, keepingthe solids concentration at 20 g/L, the inlet temperature at 200° C. andoutlet temperature at 69° C. Water still condensed in the collectionvessel and the powder looked wet. It was concluded that calcium chlorideis too hygroscopic to be spray dried without mixing with other salts orwith an excipient to reduce the calcium chloride content in the finalpowder.

Pure magnesium chloride was spray dried in the Labplant system with aninlet temperature of 195° C. and outlet temperature of 68° C. The liquidfeed consisted of 20 g/L solids concentration of magnesium chloridehexahydrate in D.I. water. The dry powder in the collection vessellooked wet and the median particle size measured on the HELOS/RODOSsystem was 21 microns. The liquid feed was then changed to 70% ethanolto reduce humidity in the exhaust drying gas, keeping the solidsconcentration at 50 g/L, the inlet temperature at 200° C. and an outlettemperature of 74° C. This magnesium chloride powder did not look wetand had a median volume particle size of 4 microns, but the powderappeared granular and had a fine particle fraction less than 5.6 micronsof 19%, indicating that the powder was not sufficiently respirable.

Example 20 Large, Porous Particles

TABLE 32 Large Porous Particle formulations x50 Spray- (μm) GSD tecFPF_TD FPF_TD % Tap @ 1 @ 1 dV50 Spraytec water <3.4 um <5.6 um Massyield density Formulation Method bar bar (μm) GSD % % % collected %(g/cc) leucine:Cacl2:NaCl 50:29.5:20.5 Niro 25.9 5.8 18.2% 29.0% 48.6%43.2% leucine:Cacl2:NaCl 50:29.5:20.5 Niro 12.2 6.3 35.4%leu:CaCl2:Na2SO4 90:4.4:5.6 Niro 10 2.4 1.8% 5.0% 16.5% 34.7% 84.8%leu:CaLact:NaCl 10:66.6:23.4 Buchi 22.4 4.4 4.9% 7.3% 13.1% 72.0% HPleu:CaCl2:Na2SO4 67.6:30:2.4 Buchi 21.2 3 13.2% 25.2% 47.7% n/a 0.22 HP

Example 21 Stability

Dry powders were tested for in-use stability under extreme temperatureand humidity conditions (ICH, Climatic Zone XIV), defined as 30° C. and75% RH. Approximately 25 mg of Formulation I, Formulation II andFormulation III were filled into capsules. The capsules were left openedand then were placed in a stability chamber at the defined conditionsfor 15 and 30 minutes. The capsules were removed at the appropriatetime, closed and tested for aerodynamic particle size distribution(aPSD) using the collapsed 2-stage ACI and for geometric particle sizedistribution (gPSD) using the Malvern Spraytec. Both tests were run at60LPM for 2 seconds. Each timepoint was repeated n=2. The results werecompared with aPSD/gPSD data from the powder at room temperature and25-30% RH.

All formulations (Formulation I, Formulation II and Formulation III)showed less than +/−5% change from the fine particle fraction of thetotal dose (FPFTD) less than 5.6 microns at standard conditions (22° C.,25-30% RH), after a 30 minute exposure to extreme temperature andhumidity conditions (30° C., 75% RH). For gPSD, Formulation I showed anincrease of approximately 30% after 30 minutes, while Formulation IIremained mostly stable and Formulation III had a decrease in Dv50 ofapproximately 15% after 30 minutes.

While insignificant changes in aerosol properties of the threeformulations were seen upon exposure to 30° C., 75% RH for 30 minutes,changes in geometric particle size were more evident (FIGS. 31A and31B). Formulation I (calcium citrate) particle size increased byapproximately 30%, while Formulation III (calcium lactate) particle sizedecreased by approximately 15%. Formulation II (calcium sulfate)particle size decreased, but not significantly.

Additional formulations tested were a calcium chloride powder (38.4%leucine, 30.0% calcium chloride, 31.6% sodium chloride) and the calciumlactate powders using different excipients (lactose, mannitol,maltodextrin) matching the Formulation III formulation (10.0% excipient,58.6% calcium lactate, 31.4% sodium chloride).

After a 30 minute exposure to extreme temperature and humidityconditions (30° C., 75% RH), the maltodextrin (Formulation IV) andmannitol formulations showed an overall change of less than +/−10%change from the fine particle fraction of the total dose smaller than5.6 microns at standard conditions (22° C., 25-30% RH). The calciumchloride powder and lactose formulation appeared affected with adecrease of over 50% and an increase of approximately 20%, respectively,in fine particle fraction of the total dose smaller than 5.6 microns.(FIG. 31C) For gPSD, the results were opposite, where the calciumchloride powder and the lactose formulation showed an overall change ofless than +/−10% change in Dv₅₀ after 30 minutes, while the mannitolformulation had an increase in Dv₅₀ of 30%-60% during the test. (FIG.31D) The maltodextrin formulation was not tested for change in Dv₅₀.

Example 22 Short-Term Stability at Room Temperature and 30% and 40% RH

Spray dried powders were kept at room temperature at approximately 30%and 40% RH for a period of one week and periodically tested for particlesize distribution. Size 3 HPMC capsules (Quali-V, Qualicaps, Whitsett,N.C.) were half filled with each dry powder. One sample was testedimmediately in the Spraytec (Malvern Instruments Inc., Westborough,Mass.), a laser diffraction spray particle sizing system where drypowders can be dispersed from an inhaler using the inhaler cell setup.Approximately 16 capsules were filled with each powder. Half of thecapsules were kept in the lab at controlled humidity and temperatureconditions (˜23-28% RH), while the other half were kept in the outsidelab at varying temperature and relative humidity (˜38-40% RH). Atspecific time points (t=1 hr, 2 hr, 4 hr, 24 hr, 48 hr, 1 week), onecapsule from the environmental controlled room and one from the outsidelab were tested on the Spraytec for volume particle size distribution.

Results for a selection of formulations containing 50% leucine and acombination of calcium chloride and the sodium salt indicated are shownin FIG. 32 and FIG. 33. The formulations containing calcium chloride andsodium chloride showed significant agglomeration after exposure tohigher humidity conditions. The acetate formulation had variable resultsat the initial time points. The sulfate, citrate and carbonateformulations demonstrated good relative stability over the test period.

Dry powder formulations containing calcium chloride and sodium chloridewere not stable when held at room temperature and 40% RH after an hourof exposure, while the acetate formulation also showed variable resultsin particle size. The sulfate and lactate powders increased slightly insize, while carbonate and citrate powders decreased slightly in size.Formulations containing only chloride and those containing acetate werenot deemed suitably stable for further study.

Example 23 Dry Powder Flow Properties

The flowability of Formulation I, II, III and IV powders was alsoassessed using conventional methods in the art for the characterizationof powder flowability. The Flowability Index for each powder wasdetermined using a Flodex Powder Flowability Test Instrument (HansonResearch Corp., model 21-101-000). For any given run, the entire samplewas loaded using a stainless steel funnel aimed at the center of thetrap door hole in the cylinder. Care was taken not to disturb the columnof powder in the cylinder. After waiting ˜30 sec for the potentialformation of flocculi, the trap door was released while causing aslittle vibration to the apparatus as possible. The test was considered apass if the powder dropped through the trap door so that the hole wasvisible looking down through the cylinder from the top and the residuein the cylinder formed an inverted cone; if the hole was not visible orthe powder fell straight through the hole without leaving a cone-shapedresidue, the test failed. Enough flow discs were tested to find theminimum size hole the powder would pass through, yielding a positivetest. The minimum-sized flow disc was tested two additional times toobtain 3 positive tests out of 3 attempts. The flowability index (FI) isreported as this minimum-sized hole diameter.

Bulk and tap densities were determined using a SOTAX Tap Density Testermodel TD2. For any given run, the entire sample was introduced to atared 100-mL graduated cylinder using a stainless steel funnel. Thepowder mass and initial volume (V₀) were recorded and the cylinder wasattached to the anvil and run according to the USP I method. For thefirst pass, the cylinder was tapped using Tap Count 1 (500 taps) and theresulting volume V_(a) was recorded. For the second pass, Tap Count 2was used (750 taps) resulting in the new volume V_(b1). If V_(b1)>98% ofV_(a), the test was complete, otherwise Tap Count 3 was used (1250 taps)iteratively until V_(bn)>98% of V_(bn−1). Calculations were made todetermine the powder bulk density (d_(B)), tap density (d_(T)), HausnerRatio (H) and Compressibility Index (C), the latter two of which arestandard measures of powder flowability. “H” is the tap density dividedby the bulk density, and “C” is 100*(1−(bulk density divided by the tapdensity)). Skeletal Density measurement was performed by MicromeriticsAnalytical Services using an Accupyc II 1340 which used a helium gasdisplacement technique to determine the volume of the powders. Theinstrument measured the volume of each sample excluding interstitialvoids in bulk powders and any open porosity in the individual particlesto which the gas had access. Internal (closed) porosity was stillincluded in the volume. The density was calculated using this measuredvolume and the sample weight which was determined using a balance. Foreach sample, the volume was measured 10 times and the skeletal density(d_(s)) was reported as the average of the 10 density calculations withstandard deviation.

Results for these density and flowability tests are shown in Tables 34and 35. All four of the powders tested possess Hausner Ratios andCompressibility Indices that are described in the art as beingcharacteristic of powders with extremely poor flow properties (See,e.g., USP <1174>). It is thus surprising that these powders are highlydispersible and possess good aerosolization properties as describedherein.

TABLE 33 Bulk and tap densities and flow properties of Formulation I-IVpowders. FI d_(B) d_(T) Sample (mm) (g/mL) (g/mL) H C Formulation I 260.193 0.341 1.77 43.4% Formulation III 22 0.313 0.722 2.31 56.7%Formulation II 18 0.177 0.388 2.19 54.3% Formulation IV >34 0.429 0.7511.75 42.9%

TABLE 34 Skeletal density measurements of powders Formulation I-IV.Sample d_(S1) ± σ (g/mL) d_(S2) ± σ (g/mL) Formulation I 1.7321 ± 0.00141.7384 ± 0.0042 Formulation III 1.6061 ± 0.0007 1.6074 ± 0.0004Formulation II 2.1243 ± 0.0011 2.1244 ± 0.0018 Formulation IV 1.6759 ±0.0005 1.6757 ± 0.0005

USP <1174> mentioned previously notes that dry powders with a HausnerRatio greater than 1.35 are poor flowing powders. Flow properties anddispersibility are both negatively effected by particle agglomeration oraggregation. It is therefore unexpected that powders with Hausner Ratiosof 1.75 to 2.31 would be highly dispersible

Example 24 Water Content and Hygroscopicity

The water content of Formulation I, II, III and IV powders wasdetermined via both thermogravimetric analysis (TGA) and Karl Fischeranalysis. Thermogravimetric analysis (TGA) was performed using a TAInstruments Q5000 IR thermogravimetric analyzer (New Castle, Del.).Sample was placed in an aluminum sample pan and inserted into the TGfurnace. The data acquisition and processing parameters are displayed oneach thermogram. Nickel and Alumel™ were used as the calibrationstandards. For TGA, the water content was determined from the loss ofmass of the samples upon heating to a temperature of 150° C. (for TGA,since the spray-drying solvent used was 100% water, it was assumed thatonly water was present as a volatile component in these powders). Arepresentative TGA thermogram for powder Formulation I is shown in FIG.34 Coulometric Karl Fischer (KF) analysis for water determination wasperformed using a Mettler Toledo DL39 KF titrator (Greifensee,Switzerland). Sample was placed in the KF titration vessel containingHydranal-Coulomat AD and mixed for 10 seconds to ensure dissolution. Thesample was then titrated by means of a generator electrode whichproduces iodine by electrochemical oxidation: 2 I−=> I₂+2e. Generally,one range-finding run and two replicates were obtained to ensurereproducibility. Summary data for powder water contents using thesemethods are shown in Table 35.

TABLE 35 Water content data for FORMUALTIONS I, II, III and IV via TGAand Karl Fischer. Water Content via Water Content via Powder TGA KarlFischer Formulation I 4.9% 3.9% Formulation III 2.0% 2.0% Formulation II5.1% 4.6% Formulation IV 2.2% 2.1%

A dynamic vapor sorption (DVS) step mode experiment was conducted tocompare the hygroscopicity and water uptake potential of Formulation I,II, III and IV powders versus raw calcium chloride dihydrate, as well asa 1:2 calcium chloride:sodium chloride control powder made viaspray-drying a formulation containing 38.4% leucine, 30.0% CaCl₂ and31.6% NaCl (it was determined that 30 wt % was the highest loading levelof calcium chloride that could be successfully incorporated into aspray-dried powder without undergoing deliquescence in the collectionvehicle immediately after spray-drying). With respect to the DVSoperating conditions, the powders were initially equilibrated at 0% RHthen exposed to 30% RH for 1 hour followed by exposure to 75% RH for 4hours. The mass % water uptake for each of the powders is shown in Table36. As can be seen in Table 36, both raw calcium chloride dihydrate andthe control powder were extremely hygroscopic, taking up approximately14 to 15% water upon exposure to 30% RH for 1 hour and taking up wellover 100% their mass in water after exposure to 75% RH. In contrast, theFormulation I, II, III and IV powders took up less than 2.5% water uponexposure to 30% RH for 1 hour and from 14% to 33% water upon exposure to75% RH for 4 hours.

TABLE 36 % Change in mass due to water uptake after (i) 30% RH hold for1 hour and (ii) 75% RH hold for 4 hours via DVS. % Change in Mass Due to% Change in Mass Due to Water Uptake after 30% Water Uptake after 75% RHPowder RH for 1 hr for 4 hrs CaCl₂*2H₂0 (raw) 13.7 146 CaCl₂—control15.3 124 Formulation I 1.68 14.7 Formulation III 1.27 28.3 FormulationII 2.45 20.8 Formulation IV 1.36 32.8

Example 25 Heat of Solution

Heats of solution were obtained upon dissolution of samples ofFormulations I through III in HBSS buffer in comparison to (i) a controlpowder comprised of 30% calcium chloride, 31.6% sodium chloride and38.4% leucine, (ii) raw calcium chloride dihydrate and (iii) rawleucine. Heats of solution were also obtained for Formulations VII andVIII using the same method.

As shown in Table 37, masses of Formulation I, II, and III powdercontaining equivalent moles of calcium ion were tested for thecalcium-containing samples. Results are shown in FIG. 35. As can be seenfrom the data shown in FIG. 35, Formulations I through III resulted insignificantly decreased heats of solution as compared to both rawcalcium chloride dihydrate and the control calcium powder. Calciumchloride dihydrate is known to possess a large exothermic heat ofsolution and to release a significant amount of heat upon contact withwater. Under certain circumstances, such as when a large quantity ofcalcium chloride dihydrate, or other salts that have a large exothermicheat of solution, are rapidly dissolved a large amount of heat isreleased that can cause burns. Thus, there are safety concernsassociated with contacting mucosal surfaces with calcium chloridedihydrate. These safety concerns can be alleviated by producing powders,such as Formulations I through III which do not have large exothermicheats of solution, and thus reduced potential for undesirable exothermiceffects.

In Table 37, Formulations I, II, III, VII, and VIII had ΔH (kcal/mol) of−6.9, −8.3, −4.3, −3.6, and −4.4. Calcium chloride dehydrate was used asa control in both experiments, i.e. the first experiment, whenFormulations I, II, and III were tested, and the second experiment, whenFormulations VII and VIII were tested. The calcium chloride dehydratehad a ΔH (kcal/mol) of −12.1 in the first experiment, and a ΔH(kcal/mol) of −8.4 in the second experiment.

Like for Formulations I, II, and III, which each had a relatively lowheat of solution compared to calcium chloride dehydrate, Formuations VIIand VIII also had a relatively low heat of solution compared to calciumchloride dehydrate. Thus, dry powder Formulations VII and VIII which donot have large exothermic heats of solution, have a reduced potentialfor undesirable exothermic effects.

TABLE 37 Heat of solution data for Formulations I-III and VII-VIII, acontrol powder containing calcium chloride, raw calcium chloridedihydrate and raw leucine. CaCl2•2H2O Leucine (experiment #1)Leu-CaCl2—NaCl Powder Average St. Dev. Average St. Dev. Average St. Dev.g 0.032 0.000 0.036 0.001 0.090 0.001 mmol* 0.244 0.001 0.242 0.0040.242 0.001 ΔT (deg. C.) 0.003 0.002 0.024 0.001 0.023 0.003 Q (cal)0.37 0.20 2.93 0.12 2.8 0.3 ΔH −1.5 0.8 −12.1 0.4 −11.7 1.4 (kcal/mol)*ΔH (kJ/mol)* −6 4 −50.5 1.6 −49 6 *mol Ca for all powders exceptleucine, which is in mol Leu Formulation I Formulation II FormulationIII Powder Average St. Dev. Average St. Dev. Average St. Dev. g 0.0770.000 0.032 0.000 0.090 0.000 mmol* 0.242 0.001 0.115 0.001 0.242 0.001ΔT (deg. C.) 0.014 0.002 0.008 0.002 0.009 0.002 Q (cal) 1.7 0.2 1.0 0.31.0 0.3 ΔH −6.9 1.0 −8.3 2.5 −4.3 1.1 (kcal/mol)* ΔH (kJ/mol)* −29 4 −3510 −18 4 *mol Ca for all powders

Example 26 In Vivo Pneumonia Model

Bacteria were prepared by growing cultures on tryptic soy agar (TSA)blood plates overnight at 37° C. plus 5% CO₂. Single colonies wereresuspended to an OD₆₀₀ ˜0.3 in sterile PBS and subsequently diluted 1:4in sterile PBS (˜2×10⁷ Colony forming units (CFU)/mL). Mice wereinfected with 50 μL of bacterial suspension (˜1×10⁶ CFU) byintratracheal instillation while under anesthesia.

C57BL6 mice were exposed to aerosolized liquid formulations in awhole-body exposure system using either a high output nebulizer or PariLC Sprint nebulizer connected to a pie chamber cage that individuallyholds up to 11 animals. Mice were treated with dry powder formulations(Table 38) 2 h before infection with S. pneumoniae. As a control,animals were exposed to a similar amount of 100% leucine powder.Twenty-four hours after infection mice were euthanized by pentobarbitalinjection and lungs were collected and homogenized in sterile PBS. Lunghomogenate samples were serially diluted in sterile PBS and plated onTSA blood agar plates. CFU were enumerated the following day.

Compared to control animals, calcium dry powder treated animalsexhibited reduced bacterial titers 24 hours after infection.Specifically, animals treated with a formulation comprised of calciumsulfate and sodium chloride (Formulation II) exhibited 5-fold lowerbacterial titers, animals treated with a formulation comprised ofcalcium citrate and sodium chloride (Formulation I) exhibited 10.4-foldlower bacterial titers, and animals treated with a formulation comprisedof calcium lactate and sodium chloride (Formulation III) exhibited5.9-fold lower bacterial titers. (FIG. 36)

TABLE 38 Formulations used to evaluate efficacy Formulation CompositionFormulation I 10.0% leucine, 35.1% calcium chloride, 54.9% sodiumcitrate (Active with 12.7% calcium ion) Formulation II 10.0% leucine,39.6% calcium chloride, 50.4% sodium sulfate (Active with 14.3% calciumion) Formulation III 10.0% leucine, 58.6% calcium lactate, 31.4% sodiumchloride (Active with 10.8% calcium ion)

The data presented herein show that divalent metal cationsalt-containing dry powders that are highly dispersible can bemanufactured and used to treat bacterial and viral infections.

Example 27 3 Month Refrigerated, Standard and Accelerated ConditionsStability Study

A 3 month physical stability study was conducted utilizingrepresentative samples of Formulations I through III filled into size 3HPMC capsules (Shionogi Qualicaps, Madrid, Spain) and placed at thefollowing conditions (i) 2-8° C. refrigerated storage, (ii) 25° C./60%RH, capsules stored under desiccant and (iii) 40° C./75% RH, capsulesstored under desiccant. FPF <5.6 and 3.4 as well as Dv50 (Spraytec) andwater content (Karl Fischer) were monitored out to a 3 month timepoint.As shown in Table 39, each of Formulations I through III displayed goodstability with respect to the assessed physical properties under each ofthese conditions.

TABLE 39 3 month stability study results for Formulations I-III.Formulation I Formulation III Spraytec Spraytec Condition Time FPF < 3.4FPF < 5.6 Dv50 Water FPF < 3.4 FPF < 5.6 Dv50 Water (° C./% RH) (mo) μmμm (μm) content um um (um) content Time zero 0 50% 63% 3.1 6% 42% 61%1.8 4% 25 C./60% RH 1 47% 68% 1.5 7% 42% 60% 2.0 4% 3 45% 68% 3.5 7% 42%61% 1.2 4% 40 C./75% RH 0.5 43% 66% 5.3 8% 39% 58% 1.8 6% 1 43% 65% 2.07% 40% 58% 3.0 4% 3 46% 68% 3.3 7% 47% 61% 1.5 4% 2-8 C. 3 46% 60% 2.45% 43% 63% 1.3 2% Formulation II Condition FPF < 3.4 FPF < 5.6 SpraytecWater (° C./% RH) um um (um) conten Time zero 55% 73% 3.1 5% 25 C./60%RH 56% 74% 3.6 6% 40 C./75% RH 57% 73% 2.4 6% 51% 67% 2.9 6% 56% 70% 3.95% 45% 64% 2.5 5% 2-8 C. 56% 76% 2.3 5%

Formulations I and III were also tested for stability with bulk powderin vials stored with dessicant. Dry powders were produced by spraydrying utilizing a Niro Mobile Minor spray dryer (GEA ProcessEngineering Inc., Columbia, Md.) with powder collection from a productfilter. Atomization of the liquid feed was performed using a SprayingSystems (Carol Stream, Ill.) two-fluid nozzle with gas cap 67147 andfluid cap 2850SS. The liquid feed was fed using gear pumps (Cole-ParmerInstrument Company, Vernon Hills, Ill.) directly into a static mixer(Charles Ross & Son Company, Hauppauge, N.Y.) immediately beforeintroduction into the two-fluid nozzle. Nitrogen was used as the dryinggas. Pressurized nitrogen or air can be used as the atomization gas feedto the two-fluid nozzle. The process gas inlet temperature was 282° C.and outlet temperature was approximately 98° C. with a liquid feedstockrate of 70 mL/min. The process gas rate was 80 kg/hr and the atomizationgas rate was set to 80 g/min. The atomizing gas rate can be set toachieve a certain gas to liquid mass ratio, which directly affects thedroplet size created. The pressure inside the drying drum was −2 “WC.Spray dried powders were collected onto a powder collection filter. Theliquid feedstock was prepared using 15 g/L solids concentrationdissolved in ultrapure water.

A 6 month physical stability study was then conducted with the powdersutilizing representative samples of Formulations I, H and III placed inbulk in 20 mL scintillation vials (Kimble, Vineland, N.J.) stored at thefollowing conditions (i) 2-8° C. refrigerated storage in a Dri-Shield3000 foil pouch (3M, Sanford, N.C.) with desiccant(MoistureBarrierBags.com; Concord, N.C.), (ii) 25° C./60% RH, kept in aDesi-Vac container (Control Company, Friendswood, Tex.) with desiccant(Fischer Scientific, Pittsburgh, Pa.) (iii) 40° C./75% RH, kept in aDesi-Vac container with desiccant.

FPF_TD (%)<5.6 μm and 3.4 μm, as well as Dv50 (Spraytec), calcium andsodium content (HPLC) and water content (Karl Fischer) were monitoredout to a 2 month timepoint for conditions (i) and (ii); and to 6 monthsfor condition (iii). As shown in Tables 41, 42 and 43, each ofFormulations I and III displayed good stability with respect to theassessed physical properties under each of these conditions.

TABLE 40 Long-term stability of Formulation I powder in bulk withdessicant. Condition Ca²⁺ Na⁺ H₂0 (° C./ Time FPF_TD FPF_TD Dv50 contentcontent content % RH) (mo) <3.4 μm <5.6 μm (μm) (wt %) (wt %) (wt %)Time zero 0 57% 68% 2.7 11.8% 13.6% 6.2% 25° C./60% 0.5 57% 67% 2.912.0% 13.8% 6.1% RH 1 57% 67% 2.0 12.2% 13.9% 6.2% 2 52% 64% 2.7 11.5%13.4% 6.3% 40° C./75% 0.5 57% 69% 2.7 11.8% 13.5% 6.3% RH 1 54% 66% 2.112.0% 13.8% 6.2% 2 52% 64% 2.7 11.8% 13.7% 6.4% 6 51% 64% 2.9 12.0%13.8% 6.5% 2-8° C. 0.5 58% 69% 2.6 12.0% 13.8% 6.2% 1 57% 68% 1.8 12.0%13.9% 6.1% 2 56% 65% 2.5 11.8% 13.7% 6.2%

TABLE 41 Long-term stability of Formulation III powder in bulk withdessicant. Condition Ca²⁺ Na⁺ H₂0 (° C./ Time FPF_TD FPF_TD Dv50 contentcontent content % RH) (mo) <3.4 μm <5.6 μm (μm) (wt %) (wt %) (wt %)Time zero 0 47% 64% 1.5 10.4% 11.6% 2.9% 25° C./ 0.5 48% 64% 2.1 10.6%11.7% 2.9% 60% RH 1 50% 64% 1.1 10.6% 12.1% 2.8% 2 45% 61% 1.3 10.5%11.7% 2.8% 40° C./ 0.5 50% 64% 1.7 10.2% 11.4% 2.7% 75% RH 1 50% 65% 1.410.5% 12.1% 2.8% 2 46% 61% 1.4 10.5% 11.6% 3.2% 6 47% 63% 1.6 10.7%11.9% 3.0% 2-8° C. 0.5 50% 65% 1.8 10.4% 11.6% 2.8% 1 50% 65% 1.2 10.8%12.3% 2.8% 2 47% 61% 1.4 10.6% 11.8% 2.9%

The liquid feedstock was prepared as a batch by dissolving leucine inultrapure water, then the calcium lactate, and finally the sodiumchloride. All chemicals were obtained from Spectrum Chemicals (Gardena,Calif.). The solution was kept agitated throughout the process until thematerials were completely dissolved in the water at room temperature.Dry powder was produced by spray drying utilizing a Niro Mobile Minorspray dryer (GEA Process Engineering Inc., Columbia, Md.) with powdercollection from a product filter. Atomization of the liquid feed wasperformed using a Spraying Systems (Carol Stream, Ill.) two-fluid nozzlewith gas cap 67147 and fluid cap 2850SS. The liquid feed was fed usinggear pumps (Cole-Parmer Instrument Company, Vernon Hills, Ill.) directlyinto a static mixer (Charles Ross & Son Company, Hauppauge, N.Y.)immediately before introduction into the two-fluid nozzle. Nitrogen wasused as the drying gas. The process gas inlet temperature was 263° C. to267° C. and outlet temperature 98° C. to 100° C. with a liquid feedstockrate of 66 mL/min. The process gas rate was 80 kg/hr and the atomizationgas rate was set to 80 g/min. The atomizing gas rate can be set toachieve a certain gas to liquid mass ratio, which directly affects thedroplet size created. The pressure inside the drying drum wasapproximately −2 “WC. Spray dried powders were collected onto a productcollection filter. The liquid feedstock was prepared using 15 g/L solidsconcentration dissolved in ultrapure water.

A 3 month physical stability study was conducted utilizingrepresentative samples of Formulation II hand-filled into size 3 HPMCcapsules (Capsugel, Greenwood, N.C.) placed in bulk in 20 mL HDPEbottles (Nolato, Trollhättan, Sweden) with desiccant (2.4 g silica gel)in the cap. The bottles were packaged in a heat sealed Dri-Shield 3000foil pouch (3M, Sanford, N.C.) and stored at the following conditions(i) 2-8° C. refrigerated, (ii) 25° C./60% RH, (iii) 40° C./75% RH.

FPF_TD (%)<5.6 μm and 3.4 μm, as well as Dv50 (Spraytec), calcium andsodium content (HPLC) and water content (Karl Fischer) were monitoredout to a 3 month timepoint for all conditions. As shown in Table 42Formulation II is susceptible to a decrease in water content caused bythe desiccant in the cap, but it still displayed good stability withrespect to the assessed physical properties under each of theseconditions.

TABLE 42 Long term stability of a Formulation III powder in HDPE bottlesin foil pouch. Condition Ca²⁺ Na⁺ H₂0 (° C./ Time FPF_TD FPF_TD Dv50content content content % RH) (mo) <3.4 μm <5.6 μm (μm) (wt %) (wt %)(wt %) Time zero 0 42% 60% 1.5 10% 12% 3.7% 25° C./60% 1 42% 59% 1.7 11%12% 1.9% RH 3 43% 61% 1.3 11% 13% 1.8% 40° C./75% 1 42% 59% 1.7 11% 12%1.8% RH 3 42% 61% 1.4 11% 13% 4.2% 5° C. 1 43% 60% 1.8 11% 12% 2.3% 344% 62% 1.3 11% 12% 2.0%

In addition, Formulation III was further tested for long term stabilitycapped in vials with desiccant. A 6 month physical stability study wasconducted utilizing representative samples of Formulation IIIhand-filled into size 3 HPMC capsules (Capsugel, Greenwood, N.C.) placedin 20 mL scintillation vials (Kimble, Vineland, N.J.) stored at thefollowing conditions (i) 2-8° C. refrigerated storage in a PE Bag(Fischer Scientific, Pittsburgh, Pa.) with a desiccant sponge (FischerScientific, Pittsburgh, Pa.) stored as hulk powder and not encapsulated,(ii) 25° C./60% RH, kept in a Desi-Vac container (Control Company,Friendswood, Tex.) with desiccant (Fischer Scientific, Pittsburgh, Pa.)and (iii) 40° C./75% RH, kept in a Desi-Vac container with desiccant.

FPF_TD (%)<5.6 μm and 3.4 μm, as well as Dv50 (Spraytec), and watercontent (Karl Fischer) were monitored out to a 6 month timepoint for allconditions. As shown in Table 43 Formulation III displayed goodstability with respect to the assessed physical properties under each ofthese conditions.

TABLE 43 Formulation III: Long term stability with dessicant. ConditionTime FPF_TD FPF_TD Dv50 H₂0 (° C./% RH) (mo) <3.4 μm <5.6 μm (μm)content (wt %) Time zero 0 42% 61% 1.8 4% 25° C./60% RH 1 42% 60% 2.0 4%3 42% 61% 1.2 4% 6 42% 61% 1.5 3% 40° C./75% RH 0.5 39% 58% 1.8 6% 1 40%58% 3.0 4% 3 47% 61% 1.5 4% 6 39% 59% 1.7 4% 2-8° C. 3 43% 63% 1.3 2% 644% 63% 1.6 2%

Example 28 Effect of Dry Powder Dose on its Efficacy Against FerretInfluenza

Dry powders comprising calcium and sodium were previously shown toreduce the severity of flu in an in vivo model of ferret flu (seeExample 11). Using this ferret flu model, the efficacy of increasingdoses of Formulation III was tested (10.0% leucine, 58.6% calciumlactate, 31.4% sodium chloride; 10.8% calcium ion). Control ferrets wereexposed to a powder comprised of 100% leucine under the same exposureconditions; in vitro this control powder had no effect on viralreplication. Several doses of Formulation III and the leucine controlpowder were aerosolized with a Palas Rotating Brush Generator 1000 solidparticle disperser (RBG, Palas GmbH, Karlsruhe, Germany), exposing theferrets (n=8 per group) in a nose-only exposure system 1 hour beforeinfection, 4 hours after infection and then twice daily (BID) for 4 days(days 1-4). The study was terminated on day 10. Nasal wash samples werecollected on days 1, 2, and 4 of the study and subcutaneous bodytemperatures and body weights were determined twice a day beginning onday 0 of the study. Body temperatures taken on days −3, −2 and −1 wereused as baseline temperatures from which to calculate the change frombaseline for each animal over the course of the study. The number ofinflammatory cells and the viral titer in nasal wash samples weredetermined. Ferrets infected with influenza typically show increases inbody temperature within 2 days of infection, drop body weight over thecourse of the study and show clinical signs of infection such aslethargy and sneezing. These changes coincide with an increase ininfluenza viral titers shed from the nasal cavity and increases in nasalinflammation.

The mean±SEM of changes in body temperatures for the control group andanimals given different doses of Formulation III are shown (FIG. 38A).Animals treated with Formulation III exhibited reduced body temperatureincreases as compared to the control-treated animal at the two peak daysof fever (Days 2 and 5). As shown in FIG. 38B, 2 days and 5 dayspost-infection Formulation III-treated animals exhibited lower bodytemperatures in a dose-responsive manner. On both days, control animalsexhibited the greatest increase in body temperature (Leu. FIG. 38B). Inaddition, the mean±SEM of changes in body temperatures of animalstreated with Formulation III were less than those for the controlanimals. Ferrets treated with the highest dose of Formulation IIIexhibited less severe weight loss (kinetics and max loss) and recoveredbody weight more quickly than the control animals (FIG. 38C). Similar tothe results with body temperature, on day 2 post-infection the bodyweight changes of control animals were much greater than that of ferretstreated with Formulation III; increasing calcium doses associated withless change in body temperatures (FIG. 38C).

Overall, the data indicated that Formulation III was able to decreasethe severity of influenza infection in ferrets in a dose-dependentmanner.

Example 29 Efficacy of Dry Powders in a Mouse Model of Asthma

Asthma is a disease characterized by recurrent attacks ofbreathlessness, wheezing, coughing and chest tightness, which vary inseverity and frequency from person to person and can become lifethreatening or fatal. Asthma is due to inflammation of the air passagesin the lungs and may be either a chronic respiratory impairment (chronicasthma) or an intermittent illness marked by episodic symptoms thatresult from a number of triggering events (intermittent asthma), like,for example environmental stimuli, allergen exposure, cold air, exerciseor emotional stress.

Studies of calcium and sodium formulations in a respiratory diseasemodel was undertaken to evaluate the effect of calcium and sodiumformulations on inflammation and, specifically, to assess whether theseformulations would further exacerbate changes in inflammation or couldinstead be safely administered therapeutically. To study the rolecalcium and sodium formulations, a mouse model of allergic asthma usingovalbumin (OVA) as an allergen, was employed. In this model, mice aresensitized to OVA over a period of two weeks and subsequently challengedvia aerosol with OVA. The subsequent challenge with OVA induces airwayinflammation and causes changes in pulmonary function. The principlechange in inflammation is an increase in the number of eosinophils inthe lungs. Similar changes in lung inflammation and pulmonary functionare observed in humans with asthma. Mice were sensitized and challengedto OVA as shown in FIG. 55.

Sensitizations were performed by intraperotineal injection of OVA plusalbumin. Challenges were performed by whole body exposure to nebulized1% OVA solution for 20 minutes. Treatments with two different doses(Low=0.24 mg Ca²⁺/kg and High=0.48 mg Ca²⁺/kg) of Formulation III (10%Leucine; 58.6% calcium lactate, 31.4% sodium chloride; 10.8% calciumion), or a 100% leucine dry powder were given 1 hour before or 4 hoursafter OVA challenge on days 27 to 29 and also performed twice on day 30.The dose was varied by changing the number of capsules used for eachexposure. Treatments were made in a whole body exposure chamber using acapsule-based dry powder inhaler system. On the final day of the study(day 31), mice were euthanized and bronchoalveolar lavages (BAL) wereperformed. The total number of cells per BAL was determined. Inaddition, the percentage and total number of macrophages,polymorphonuclear cells (neutrophils), lymphocytes, and eosinophils weredetermined by differential staining. Data depict the mean±SD of 4-5 miceper group and are representative of at least two different studies.

Surprisingly, treatment of mice with 0.48 mg Ca²⁺/kg reduced total BALcell counts and the number of eosinophils in the BAL to statisticallysignificant levels compared to the control animals (one-way ANOVA;Tukey's multiple comparison tests) (FIGS. 39A and 39B). Similarly, thelower dose of Formulation III (0.24 mg Ca²⁺/kg) significantly reducedeosinophils counts, but not to the same degree as the higher dose (0.48mg Ca²⁺/kg). Thus, treatment of mice with dry powder formulationscomprised of calcium lactate and sodium chloride reduced airwayinflammation in a dose responsive manner.

Thus, it was discovered that the dry powder formulations of the presentinvention, far from exacerbating inflammation, actually reduced airwayinflammation and reduced the degree of eosinophilia. This result cannotbe explained simply by the biophysical mechanism of action, since it waspreviously observed that soluble factors, such as Der p 1 or OVA, areuninhibited in their movement across mucus-like materials followingcalcium exposure. Accordingly, this discovery demonstrates that the saltformulations of the present invention have an unexpectedanti-inflammatory property and can either serve as a stand-alone therapyor be used in combination with other asthma medications for therapy ofasthma or asthma-associated symptoms.

Example 30 Efficacy of Dry Powders in a Mouse Model of COPD

Chronic obstructive pulmonary disease (COPD) is a progressive diseaseassociated with impaired pulmonary function and it primarily occurs as aresult of cigarette smoking. COPD subjects are further susceptible toexacerbations that are often associated with an infectious agent andacute inflammation. These exacerbations lead to further declines in lungfunction, which in turn drives the increased frequency and severity ofsubsequent exacerbations.

To study both the disease and potential treatments, animal models ofCOPD have been developed. Animal models of tobacco smoke (TS) exposurehave been established to facilitate the testing of novel therapeuticsand to evaluate acute airway inflammation following TS exposure (Churg,A. et al. Am J. Physiol Lung Cell Mol Physiol 294(4):L612-631, 2008;Churg, A. and J. L. Wright, Proc Am Thorac Soc 6(6):550-552, 2009; Fox,J. C. and Fitzgerald M. R., Curr Opin Pharmacol 9(3):231-242, 2009).

Accordingly, a study was performed to evaluate the efficacy of a drypowder formulation comprised of calcium and sodium salts on thepulmonary inflammation induced by TS exposure. The 4-day TS exposuremodel shown in FIG. 55B was employed.

Mice (C57BL6/J) were exposed to TS for up to 45 minutes per day on foursuccessive days by whole body exposure. On each day of TS exposure, micewere treated with Formulation III (10.0% leucine, 58.6% calcium lactate,31.4% sodium chloride; 10.8% calcium ion) 1 hour before and 6 hoursafter Ts exposure. Formulation III dosing was performed using a wholebody exposure system and a capsule based delivery system. A dry powderof 100% leucine was used as a control powder. A schematic depiction ofthe study design and the estimated doses delivered are shown below andin Table 44. A p38 MAP kinase inhibitor ADS110836 was used as areference agent (WO2009/098612 Example 11) and was administered by anintranasal route.

TABLE 44 TS mouse dry powder dosing. Group TS/Sham μg Ca/kg* No.Exposure Compound n= (per dose) Capsules 1 Sham 100% Leucine 10 — 6 2 TS100% Leucine 10 — 6 3 TS Formulation III 10 170 1 4 TS Formulation III10 700 3 5 TS Formulation III 10 1680  6 6 TS Reference p38 10  100** 0inhibitor

Different doses of calcium were delivered by increasing the number ofcapsules used. Doses were calculated by collecting samples from the piecage system onto a glass fiber filter at 1LPM. The aerosol collectedonto the filter was recovered and the calcium concentration wasdetermined by HPLC. This data was used to calculate the aerosolconcentration (E_(c)) of calcium ion, which was subsequently used todetermine the estimated dose level. The estimated dose level (D_(L)) isgiven by the equation: D_(L)=E_(c)·RMV·T/BW, where RMV is therespiratory minute volume of the animal (0.21 LPM), T is the exposuretime, and BW is the body weight of the animal in kg. The resultingestimated dose is then adjusted for the respirable fraction of theaerosol, which is determined based on the fine particle fraction (FPF; %mass less than 5.6 μm).

Animals were euthanized by intra-peritoneal barbiturate anaestheticoverdose 24 hours after the final exposure to either air (sham) or TS onday 5. A bronchoalveolar lavage (BAL) was performed using 0.4 mL ofphosphate buffered saline (PBS). Cells recovered from the BAL wereenumerated and differential cell counts carried out using cytospinprepared slides. Inflammatory cell counts in the BAL fluid of animalsexposed to TS for 4 days were determined. TS exposed animals were thenexposed to Formulation III or a control dry powder of 100% leucine. Theleucine treated animals exposed to TS exhibited a 9-fold increase intotal cell counts compared to air treated animals that were alsoadministered the control powder (FIG. 40A). The magnitude of thisincrease demonstrated the degree of inflammation observed after 4-daysof TS exposure. Additional groups of animals were exposed to increasingdoses of calcium-containing dry powder. Increasing doses were achievedby increasing the number of capsules used for each exposure.

As shown in FIG. 40A, total cell counts in the BAL fluid were reduced byFormulation III treatment in a dose-responsive manner compared to thecontrol group (14% reduction for the low dose, 32% reduction for the middose, and 45% reduction for the high dose). At the highest dose tested,the reduction was comparable to that of the positive control p38 MAPKinhibitor treatment (51% reduction). In addition, Formulation IIIsignificantly reduced the number of macrophages (FIG. 40B), neutrophils(FIG. 40C) and lymphocytes (FIG. 40D), in the BAL samples, with thegreatest percent reduction observed for neutrophils and lymphocytes. Ofnote, even the low dose of calcium (1 capsule) reduced neutrophils whilelymphocytes to statistically significant levels and the high dosereduced these cell types to levels that were comparable to the positivecontrol compound (p38 inhibitor).

Together, the data indicated that aerosol delivery of dry powderformulations comprised of calcium and sodium salts can effectively limitinflammation and have a general anti-inflammatory effect. The magnitudeof the effect is comparable to other drugs that are known to beeffective in the model. The data suggested that dry powder formulationscomprised of calcium and sodium salts could be used to treat COPD and,further, that a combination with other drugs used for the treatment ofCOPD (e.g., ICS, bronchodiolators (LABA/LAMA), p38 MAPK inhibitors, PDE4inhibitors, antibody therapies, NF-κB inhibitors, and the like) wouldprovide an enhanced benefit. To determine the specific cell types thatwere reduced by the treatment, differential cell counts of the same BALsamples were performed. Of note, the inflammation characteristic of themodel is marked by increases in macrophages and neutrophils, with modestincreases observed in lymphocytes and epithelial cells.

Example 31 Stability of Dry Powder Formulations with Various Excipients

Dry powders comprised of calcium lactate and sodium chloride and furthercomprising other excipients (e.g., maltodextrin and mannitol) weretested for their stability as discussed previously (Example 37). Thecompositions of these formulations can be found in Table 45.

TABLE 45 Dry powder calcium and sodium formulations. FormulationCompositions Ca:Na % % Calcium % Sodium % % molar Excipient Calcium saltSodium salt Ca²⁺ Na⁺ Form ratio Excipient (w/w) salt (w/w) salt (w/w)(w/w) (w/w) III 1:2 Leucine 10.0 CaLac₂ 58.6 NaCl 31.4 10.8 12.4 IV 1:2Malto- 10.0 CaLac₂ 58.6 NaCl 31.4 10.8 12.4 dextrin  V 1:2 Mannitol 10.0CaLac₂ 58.6 NaCl 31.4 10.8 12.4

The dry powders were made from liquid feedstock that was prepared as ahatch by dissolving the excipient (mannitol or maltodextrin) inultrapure water, then the calcium lactate, and finally the sodiumchloride. All chemicals were obtained from Spectrum Chemicals (Gardena,Calif.). The solution was kept agitated throughout the process until thematerials were completely dissolved in the water at room temperature.The solids concentration was 5 g/L in ultrapure water.

Formulation IV and V dry powders were produced by spray drying on theBüchi B-290 Mini Spray Dryer (BÜCHI Labortechnik AG, Flawil,Switzerland) with powder collection from a High Performance cyclone on aglass vessel with a plastic cover. The system used the Büchi B-296dehumidifier. Atomization of the liquid feed utilized a Büchi two-fluidnozzle with a 1.5 mm diameter. The two-fluid atomizing gas was set at 40mm and the aspirator rate to 90%. Room air was used as the drying gas.Inlet temperature of the process gas was 220° C. and outlet temperatureat 99° C. to 104° C. with a liquid feedstock flow rate of 5 mL/min to 6mL/min.

A 6 month physical stability study was subsequently conducted utilizingrepresentative samples of Formulation IV and V hand-filled into size 3HPMC capsules (Capsugel, Greenwood, N.C.) or kept in bulk, both placedin 20 mL scintillation vials (Kimble, Vineland, N.J.) stored at thefollowing conditions (i) 2-8° C. bulk refrigerated, storage in a PE Bag(Fischer Scientific, Pittsburgh, Pa.) with a desiccant sponge (FischerScientific, Pittsburgh, Pa.), (ii) 25° C./60% RH, capsules kept in aDesi-Vac container (Control Company, Friendswood, Tex.) with desiccant(Fischer Scientific, Pittsburgh, Pa.) (iii) 40° C./75% RH, capsules keptin a Desi-Vac container with desiccant.

FPF_TD (%)<5.6 μm and 3.4 μm, as well as Dv50 (Spraytec), and watercontent (Karl Fischer) were monitored out to a 6 month timepoint for allconditions. As shown in Table 46 Formulation IV and V both displayed anincrease in FPF_TD (%)<5.6 μm and 3.4 μm; however, the change was lessthan 20% from the values at time zero. Formulation IV showed goodstability with respect to Dv50, while Formulation V showed an increaseof over 20% in Dv50 for conditions (ii) and (iii). Formulation V showedgood stability in water content for conditions (ii) and (iii) and adecrease of over 20% in water content for condition (i). Formulation IValso presented a decrease in water content of over 20% for conditions(i) and (ii), with stable water content for condition (iii). Theseresults suggested that Formulation IV and V were sensitive to a decreasein water content when stored with desiccant. The decrease in watercontent could be the root cause for the decrease in particle size seenin the cascade impaction results.

TABLE 46 Stability of dry powders containing maltodextrin and mannitol.Formulation IV Formulation V FPF_TD FPF_TD H₂0 FPF_TD FPF_TD H₂0Condition Time <3.4 <5.6 50 content <3.4 <5.6 50 content (° C./% RH)(mo) μm μm (μm) (wt %) μm μm (μm) (wt %) Time zero 0 45% 67% 1.6 6.7%45% 66% 1.8 5.3% 25° C./60% RH 1 43% 63% 1.7 5.3% 45% 63% 1.7 5.3%(capsules/vial/ 3 49% 71% 1.4 4.9% 49% 70% 1.4 4.9% dessicator) 6 49%74% 2.2 4.5% 54% 77% 1.6 4.4% 40° C./75% RH 0.5 47% 68% 1.8 6.8% 45% 62%1.9 5.6% (capsules/vial/ 1 46% 67% 2.0 5.1% 48% 66% 1.9 4.9% desiccator)3 52% 74% 2.7 5.4% 53% 75% 1.3 4.9% 6 50% 71% 1.5  26% 53% 73% 1.6 5.2%2-8° C. 3 50% 71% 1.8 4.7% 50% 73% 1.6 3.8% (bulk/vial) 6 52% 74% 2.04.5% 56% 77% 1.7 5.8%

Example 32 Dispersibility of Dry Powder Formulations Containing Leucine,Mannitol or Maltodextrin

The dispersibility of dry powder Formulations III, IV and V was assessedas described previously (see Example 15) by measuring the geometricparticle size and the percentage of powder emitted from capsules withinhalation on a dry powder inhaler using flow rates representative ofpatient use. The particle size distribution and weight change of thefilled capsules were measured as a function of flow rate, inhaledvolume, and fill weight in a passive dry powder inhaler. At each flowrate, the mass of powder emitted from 5 replicate capsules was measuredand the Dv(50) and CEPM results were averaged.

FIG. 41A shows the dose emitted from capsules containing FormulationsIII, IV or V at a 50 mg fill weight in a passive high resistance drypowder inhaler (RS-01 Model 7, High Resistance, Plastiape S.p.A., 0.036kPa^(1/2)LPM⁻¹ resistance). As can been in FIG. 41A, all three powdersemptied well from the capsules. Formulations IV and V had a similar CEPMas a function of inhalation energy, with greater than 90% of the powdersemitting at 15-20 LPM, while Formulation III had about 90% CEPM at 25LPM. FIG. 41B shows the particle size distribution of Formulations III,IV and V when emitted from the inhaler as characterized by the volumemedian diameter, Dv(50), plotted against the inhalation energy applied.Consistent with their CEPMs, Formulations IV and V also had similarDv(50) characteristics while Formulation III had a higher particle sizea lower flow rates (FIG. 41B). Overall, the Dv(50) values for the drypowder formulations indicated that the powders did not agglomerate and,instead, were well-dispersed.

Example 33 Efficacy of Salt Formulations Containing Leucine, Mannitol orMaltodextrin in a Mouse Model of Bacterial Pneumonia

The anti-infective efficacy of calcium and sodium-containing dry powderswas tested in an in vivo mouse model of bacterial pneumonia (see Example26). Two-piece gel capsules (Capsugel Vcaps™, size 00CS) were filled tothe appropriate weight with dry powders containing calcium lactate,sodium chloride and either leucine (Formulation III), maltodextrin(Formulation IV) or mannitol (Formulation V). Filled capsules werestored in laboratory desiccators until the time of treatments.

Serotype 3 Streptococcus pneumoniae bacteria were prepared by growingcultures on tryptic soy agar (TSA) blood plates overnight at 37° C. plus5% CO₂. Single colonies were re-suspended in sterile PBS to an opticaldensity at 600 nm (OD₆₀₀) of 0.3 in sterile PBS and subsequently diluted1:2 in sterile PBS [˜4×10⁷ Colony forming units (CFU)/mL].

C57BL6 mice were treated with either a leucine dry powder or theaforementioned dry powder (0.24 mg/kg Ca dose) formulations for 2.5minutes/capsule in a whole-body exposure system. Dry powder aerosol wasgenerated using a capsule based delivery system connected to atop-loading pie chamber cage that individually holds up to 11 animals.All dry powder treatments were delivered at 10 psi and 7 scfh (≈2.8L/min). Dry powder treatments were performed 2 hours before mice wereinfected with 50 μL of S. pneumoniae suspension (˜2×10⁶ CFU) byintratracheal instillation while under anesthesia. Twenty-four hoursafter infection, mice were euthanized by pentobarbital injection andlungs were collected and homogenized in sterile PBS. Lung homogenatesamples were serially diluted in sterile PBS and plated on TSA bloodagar plates. Agar plates were incubated overnight at 37° C. and CFU wereenumerated the following day for quantification of bacterial burden inthe lungs.

The lung bacterial burden in each animal is shown in FIG. 42. Eachcircle represents data from a single animal and the bar depicts thegeometric mean for the group. Data were normalized to the leucinecontrol in each respective experiment. Data are pooled from twoindependent experiments. The treatment groups were compared to theleucine control group by two-tailed Student 1-test. Compared to controlanimals, mice treated with all three dry powder formulations exhibitedreduced bacterial titers 24 hours after infection. Animals treated witha formulation comprised of calcium lactate, sodium chloride and leucine(Formulation III) exhibited 5.9-fold lower bacterial titers, while thosetreated with mannitol- and maltodextran-containing powders exhibitedapproximately 2-fold lower bacterial burden. These data indicated thatalthough calcium-sodium dry powders comprising leucine, maltodextran andmannitol were all effective, that leucine-containing powder (FormulationIII) were the most effective at treating bacterial infection.

Example 34 Efficacy of Dry Powders Containing Leucine, Mannitol orMaltodextrin in a Mouse OVA Model of Allergic Asthma

Calcium and sodium formulations also comprising either leucine,mannitol, or maltodextrin were further evaluated for their ability toinhibit the inflammatory cell response associated with allergic asthma.The powders were tested in the OVA mouse model described previously (seeExample 29). Briefly, mice were sensitized to, then challenged with OVAto induce airway inflammation similar to that seen in humans withasthma. Mice were treated with leucine alone or Formulation III,Formulation IV or Formulation V by whole body exposure 1 hour before or4 hours after OVA challenge on days 27 through 29 and twice on day 30.On day 31, BALs were performed and total number of cells and eosinophilsfrom them were determined by differential staining. Data depict thestandard deviation of 4-5 mice per group and are representative of atleast two different studies. Although cell counts in the leucine controlwere somewhat low, it is clear that mice treated with Formulation IIIhad much lower total (FIG. 43A) and eosinophil (FIG. 43B) cell countsthan those treated with Formulation IV or Formulation V, indicating thatcalcium and sodium dry powders comprising leucine were the mosteffective in inhibiting asthma-associated inflammation.

Example 35 Characteristics of Powders with Various Amounts of Leucineand Different Molar Ratios of Calcium to Sodium Ion

Components of dry powder salt formulation can affect both theirstability and their efficacy. In order to ascertain the effect ofincreasing levels of leucine, and that of increasing the molar ratio ofcalcium ion to sodium ion on the dry powders, dry powders formulationswere produced.

The liquid feedstock for the powders was prepared as a batch bydissolving leucine in ultrapure water, then the calcium lactate, andfinally the sodium chloride. All chemicals were obtained from SpectrumChemicals (Gardena, Calif.). The solution was kept agitated throughoutthe process until the materials were completely dissolved in the waterat room temperature.

Formulation III was prepared as described previously (see Example 27)and was from the same lot. All other powders were produced by spraydrying on the Büchi B-290 Mini Spray Dryer (BÜCHI Labortechnik AG,Flawil, Switzerland) with powder collection from a High Performancecyclone on a 60 mL glass vessel. The system used the Büchi B-296dehumidifier. Furthermore, when the relative humidity in the roomexceeded 30% RH, an external LG dehumidifier (model 49007903, LGElectronics, Englewood Cliffs, N.J.) was run constantly. Atomization ofthe liquid feed utilized a Büchi two-fluid nozzle with a 1.5 mmdiameter. The two-fluid atomizing gas was set at 40 mm and the aspiratorrate to 90%. Room air was used as the drying gas. Inlet temperature ofthe process gas was 220° C. and outlet temperature at 94° C. to 102° C.with a liquid feedstock flow rate of 4.9 mL/min to 5.3 ml/min. Thesolids concentration was 10 g/L in dissolved in ultrapure water.

The powders produced were characterized (e.g., for size, water content)as described previously, and the results of these characterizations areshown in Table 47.

TABLE 47 Characteristics of dry powders comprising various amounts ofleucine and different molar ratios of calcium to sodium ion. FormulationSpraytec ACI-2 Other H₂0 Ca:Na Dv(50) GSD FPF_TD FPF_TD content YieldConc ratio Leu CaLact NaCl (μm) (μm) <3.4 μm <5.6 μm (wt %) % (g/L) 1:210.0 58.6 31.4 1.5 2.5 44.6% 62.8% 2.7% 88.4% 15 8:1 10.0 87.1 2.9 4.23.5   26% 57.5% 2.3% 78.8% 10 20.0 77.4 2.6 4.5 3.3 28.9% 57.1% 2.6%67.2% 10 30.0 67.7 2.3 5.2 3.9 32.6% 58.6% 71.6% 10 39.4 58.6 2.0 5.73.6 36.2% 60.8% 2.0% 76.3% 10 69.0 30.0 1.0 7.3 3.8 49.0% 71.3% 1.7%56.9% 5 2:1 10.0 79.4 10.6 4.5 3.5 31.1% 55.5% 2.5% 67.2% 10 20.0 70.69.4 1.8 2.8 43.4% 65.0% 3.4% 68.6% 5 30.0 61.7 8.3 3.7 3.4 40.9% 67.0%3.3% 83.2% 5 33.6 58.6 7.8 3.6 3.3 33.5% 55.7% 3.2% 69.8% 10 66.0 30.04.0 5.3 3.8 51.3% 73.1% 2.1% 63.7% 5

Example 36 Stability of Dry Powders Comprising Calcium Lactate, SodiumChloride and Leucine at Different Ratios

The stability of several of the powders produced was then evaluatedunder conditions described previously. A 2 month physical stabilitystudy was conducted utilizing representative samples of Formulation III,VI and three more formulations encompassing a range of leucine loadingand Ca:Na molar ion ratio of 2:1 and 8:1. The dry powders werehand-filled into size 3 HPMC capsules (Capsugel, Greenwood, N.C.) andplaced in 20 mL scintillation vials (Kimble, Vineland, N.J.) and heatsealed in a Dri-Shield 3000 foil pouch (3M, Sanford, N.C.) stored at thefollowing conditions (i) 2-8° C., (ii) 25° C./60% RH and (iii) 40°C./75% RH.

FPF_TD (%)<5.6 μm and 3.4 μm, as well as Dv50 (Spraytec), calcium andsodium content (HPLC) and water content (Karl Fischer) were monitoredout to a 2 month timepoint for all conditions. As shown in Table 48, allformulations displayed good stability with respect to the assessedphysical properties under each of these conditions.

TABLE 48 Stability of salt formulations comprising various amounts ofleucine and different molar ratios of calcium to sodium ion. H₂0 H₂0Condition Time FPF_TD FPF_TD Dv50 content FPF_TD FPF_TD Dv50 content (°C./% RH) (mo) <3.4 μm <5.6 μm (μm) (wt %) <3.4 μm <5.6 μm (μm) (wt %)Formulation III 33.6% Leucine Ca:Na 2:1 Time zero 0 44% 65% 1.5 3.6% 31%58% 3.6 4.6% 25° C./60% RH 1 45% 65% 1.0 3.9% 35% 59% 2.6 4.7% 2 45% 63%1.3 4.1% 31% 56% 4.0 4.7% 40° C./75% RH 1 45% 64% 1.2 4.1% 26% 52% 3.84.6% 2 45% 63% 1.3 4.0% 26% 52% 5.7 4.4% 5° C. 1 45% 64% 1.5 3.4% 32%56% 3.1 4.4% 2 44% 60% 1.7 3.5% 32% 57% 4.6 4.4% 10% Leucine Ca:Na 8:120% Leucine Ca:Na 8:1 Time zero 0 29% 52% 3.7 4.4% 29% 51% 5.2 3.8% 25°C./60% RH 1 32% 54% 2.4 4.9% 30% 52% 3.1 4.6% 2 35% 58% 3.9 5.0% 31% 52%4.4 4.5% 40° C./75% RH 1 33% 55% 0.9 5.1% 32% 54% 3.5 4.9% 2 32% 54% 4.15.0% 29% 50% 3.2 4.8% 5° C. 1 31% 52% 2.9 4.4% 28% 51% 3.3 4.0% 2 31%54% 4.0 4.5% 33% 56% 5.1 4.1% 39.4% Leucine Ca:Na 8:1 Time zero 0 33%57% 4.8 3.5% 25° C./60% RH 1 35% 60% 4.3 3.8% 2 35% 59% 4.7 3.8% 40°C./75% RH 1 31% 55% 4.9 4.0% 2 33% 56% 4.8 3.9% 5° C. 1 33% 56% 5.6 3.3%2 35% 58% 6.1 3.3%

Example 37 Aerosol Properties of Dry Powders with Different Amounts ofLeucine and Various Calcium to Sodium Ion Molar Ratios

Dry powders were further evaluated for their dispersibility. The drypowders tested are shown in Table 49.

TABLE 49 Dry powder calcium and sodium formulations. FormulationCompositions % % Ca:Na % Calcium Sodium % % molar Excipient Calcium saltSodium salt Ca²⁺ Na⁺ Form ratio Excipient (w/w) salt (w/w) salt (w/w)(w/w) (w/w) III 1:2 Leucine 10.0 CaLac₂ 58.6 NaCl 31.4 10.8 12.4 VI 8:1Leucine 39.4 CaLac₂ 58.6 NaCl 2.0 10.8 0.8 VII 4:1 Leucine 37.5 CaLac₂58.6 NaCl 3.9 10.8 1.5 VIII 4:1 Leucine 20.0 CaLac₂ 75.0 NaCl 5.0 13.82.0

The dispersibility of the above dry powder formulations were assessedwhen delivered from a dry powder inhaler over a range of inhalation flowrates and volumes. This dispersibility was investigated by measuring thegeometric particle size and the percentage of powder emitted fromcapsules when inhaling on a dry powder inhaler with flow ratesrepresentative of patient use. The particle size distribution and weightchange of the filled capsules were measured for multiple powderformulations as a function of flow rate, inhaled volume and fill weightin a passive dry powder inhaler.

Powder formulations were filled into size 3 HPMC capsules (CapsugelV-Caps) by hand with the fill weight measured gravimetrically using ananalytical balance (Mettler Toledo XS205). Fill weights of 50 mg werefilled for Formulations III, VI, VII, and VIII. A capsule-based passivedry powder inhaler (RS-01 Model 7, High Resistance, Plastiape S.p.A.)was used which had specific resistances of 0.036 kPa^(1/2)LPM⁻¹. Flowrate and inhaled volume were set using a timer controlled solenoid valvewith flow control valve with an inline mass flow meter (TSI model 3063).Capsules were placed in the dry powder inhaler, punctured and theinhaler sealed inside a cylinder, exposing the outlet of the DPI to thelaser diffraction particle sizer (Spraytec, Malvern) in its open benchconfiguration. The steady air flow rate through the system was initiatedusing the solenoid valve and the particle size distribution was measuredvia the Spraytec at 1 kHz for the duration of the single inhalationmaneuver with a minimum of 2 seconds. Particle size distributionparameters calculated included the volume median diameter (Dv50), thegeometric standard deviation (GSD), and the fine particle fraction (FPF)of particles less than 5 micrometers in diameter. At the completion ofthe inhalation duration, the dry powder inhaler was opened, the capsuleremoved and re-weighed to calculate the mass of powder that had beenemitted from the capsule during the inhalation duration. At each testingcondition, 5 replicate capsules were measured and the results of Dv50,FPF and capsule emitted powder mass (CEPM) were averaged.

In order to relate the dispersion of powder at different flow rates,volumes, and from inhalers of different resistances, the energy requiredto perform the inhalation maneuver was calculated and the particle sizeand dose emission data plotted against the inhalation energy. Inhalationenergy was calculated as E=R²Q²V where E is the inhalation energy inJoules, R is the inhaler resistance in kPa^(1/2)/LPM, Q is the steadyflow rate in L/min and V is the inhaled air volume in L.

FIG. 44 shows the dose emitted from a capsule for Formulations III, VI,VII and VIII at a capsule fill weight of 50 mg using the high resistanceRS-01 dry powder inhaler. For each powder, a 2 L inhalation was used atthe high flow rate condition of 60 LPM, corresponding to the highestenergy condition of 9.2 Joules. For the other three flow rates of 30, 20and 15 LPM, an inhalation volume of 1 L was used. As can be seen fromFIG. 44, the entire mass of powder filled into the capsule emptied outof the capsule in a single inhalation for all 4 formulations at thehighest energy condition tested. For Formulation III, greater than 80%of the fill weight emptied for all tested inhalation conditions. ForFormulations VI and VIII, capsule dose emission dropped below 80% of thefill weight at 0.29 Joules. For Formulation VII, capsule dose emissiondropped below 80% of the fill weight at 0.51 Joules.

The particle size distributions of the emitted powder of FormulationsIII, VI, VII and VIII are listed in the Table 50, as characterized bythe Dv50 and GSD as a function of the applied flow rate and inhalationenergy. Consistent values of Dv50 at decreasing energy values indicatethat the powder is well-dispersed since additional energy does notresult in additional deagglomeration of the emitted powder. The Dv50values were consistent for all 4 Formulations with the mean Dv50increasing by less than 2 micrometers from the highest inhalation energycondition (and hence most dispersed state) down to inhalation energiesof 0.29 Joules. For Formulation VIII, the mean Dv50 did not increasefrom baseline by 2 micrometers over the whole tested range with themaximum increase of 1.4 micrometers (from 2.1 to 3.5 micrometers) for adecrease of inhalation energy from 9.2 Joules to 0.29 Joules. In theseranges, the Dv50 was not significantly increased in size, which would beexpected if the emitting powder contained a lot of agglomerates and wasnot well dispersed.

TABLE 50 Particle size distribution of emitted dry powders. InhaledEnergy (J), E = R²Q²V 9.2 1.1 0.5 0.3 Flow Rate (LPM) 60 30 20 15Formulation Dv50 (μm) 1.0 ± 0.1 1.4 ± 0.1 2.3 ± 0.2 3.1 ± 0.3 III GSD6.0 ± 0.4 4.4 ± 0.3 3.7 ± 0.6 3.4 ± 0.7 FPF < 5 μm 85.2 ± 1.1  85.5 ±0.7  78.0 ± 1.1  68.1 ± 1.9  Formulation Dv50 (μm) 3.3 ± 0.2 4.0 ± 0.25.2 ± 0.2 6.2 ± 0.7 VI GSD 5.5 ± 0.4 4.6 ± 0.5 4.4 ± 0.3 3.3 ± 0.2 FPF <5 μm 61.4 ± 1.4  57.1 ± 1.4  48.7 ± 1.2  41.5 ± 4.2  Formulation Dv50(μm) 2.0 ± 0.2 3.0 ± 0.2 3.6 ± 0.1 5.0 ± 0.3 VII GSD 5.6 ± 0.2 4.3 ± 1.03.7 ± 0.5 3.6 ± 0.2 FPF < 5 μm 69.5 ± 0.8  64.9 ± 2.3  62.0 ± 1.7  49.7± 2.3  Formulation Dv50 (μm) 2.1 ± 0.4 2.1 ± 0.1 2.8 ± 0.1 3.5 ± 0.1VIII GSD 5.2 ± 0.3 4.3 ± 0.2 3.3 ± 0.3 3.3 ± 0.3 FPF < 5 μm 73.9 ± 1.8 74.4 ± 0.5  71.0 ± 1.2  63.2 ± 0.8 

Also assessed was the aerodynamic size distribution of the thy powderformulations when delivered from a dry powder inhaler in a rangeappropriate for deposition in the respiratory tract. The aerodynamicparticle size distributions of the four powder formulations weremeasured by characterizing the powders with an eight stage Andersoncascade impactor (ACI). Dry powder formulations were filled into size 3HPMC capsules (Capsugel V-Caps) by hand with the fill weight measuredgravimetrically using an analytical balance (Mettler Toledo XS205). Fillweights of 50 mg were filled for Formulations III, VI, and VII, and afill weights of 40 mg were filled for Formulation VIII. A reloadable,capsule based passive dry powder inhaler (RS-01 Model 7, HighResistance, Plastiape, Osnago, Italy) was used to disperse the powderinto the cascade impactor. Two capsules were used for each measurement,with two actuations of 2 L of air at 60 LPM drawn through the dry powderinhaler (DPI) for each capsule. The flow rate and inhaled volume wereset using a timer controlled solenoid valve with flow control valve(TPK2000 Copley Scientific). Three replicate ACI measurements wereperformed for Formulations VII and VIII and five replicates forFormulation VI and Eight replicates for Formulation III. The impactorstages, induction port (IP), entrance cone (EC) and after filter (F)were rinsed with measured volumes of water and the rinse solutionsassayed by HPLC for calcium ion concentration. For Formulation III, theentrance cone was not rinsed. The size distribution, MMAD, GSD and fineparticle dose <4.4 micrometers (FPD<4.4 μm) of the emitted powder wasaveraged across the replicates and are tabulated in Table 51. ForFormulations III, VI and VII, the dose filled was two capsules of 50 mgpowder fill weight which corresponded to 10.8 mg of Ca²⁺ filled into thecapsules. For Formulation VIII, the two capsules of 40 mg of powderfilled contained the same 10.8 mg of Ca²⁺ due to the formulation'shigher Ca²⁺ content.

All four formulations were found to have repeatable size distributionsas illustrated by the low standard deviations for all the tabulatedvalues (Table 51). All replicates of all four formulations had greaterthan 85% of the Ca²⁺ which was filled into the two capsules recovered inthe cascade impactor. This both shows that the dosing of theformulations from the DPI was consistent and had low and consistentpowder retention in the capsules and DPI as well as indicating that themeasured size distributions were characteristic of the full dosedelivered and not just a sample of the dose. All four formulations haverespirable doses as indicated in this test by the fine particle dose<4.4 micrometers that are a significant portion of the filled dose, withfine particle doses ranging from 2.0 mg to 5.4 mg of the filled 10.8 mgof calcium. With a maximum GSD of 2.1 for the four formulations, thepolydispersity of the size distributions was relatively small relativeto typical dry powder formulations for inhalation.

TABLE 51 Aerodynamic particle size distribution of Formulations III, VI,VII and VIII. Formulation Formulation Formulation Formulation ACI StageVIII VII VI III IP (+EC) (mg Ca²⁺) 2.64 ± 0.06 1.89 ± 0.17 2.35 ± 0.211.76 ± 0.12 −1 (mg Ca²⁺) 1.27 ± 0.14 2.06 ± 0.35 2.70 ± 0.20 0.40 ± 0.04−0 (mg Ca²⁺) 1.31 ± 0.04 2.01 ± 0.10 1.85 ± 0.07 0.62 ± 0.07 1 (mg Ca²⁺)1.31 ± 0.07 1.66 ± 0.08 1.36 ± 0.10 1.17 ± 0.12 2 (mg Ca²⁺) 0.88 ± 0.080.86 ± 0.01 0.71 ± 0.11 1.34 ± 0.10 3 (mg Ca²⁺) 1.03 ± 0.09 0.86 ± 0.080.67 ± 0.07 1.98 ± 0.15 4 (mg Ca²⁺) 0.56 ± 0.06 0.52 ± 0.03 0.37 ± 0.061.26 ± 0.14 5 (mg Ca²⁺) 0.22 ± 0.01 0.27 ± 0.03 0.17 ± 0.02 0.49 ± 0.056 (mg Ca²⁺) 0.09 ± 0.01 0.09 ± 0.01 0.02 ± 0.03 0.14 ± 0.03 F (mg Ca²⁺)0.10 ± 0.03 0.14 ± 0.01 0.07 ± 0.01 0.22 ± 0.03 FPD < 4.4 (mg Ca²⁺) 2.88± 0.10 2.72 ± 0.08 2.01 ± 0.18 5.43 ± 0.29 μm MMAD (μm) 5.22 ± 0.21 6.29± 0.32 7.17 ± 0.23 3.12 ± 0.11 GSD 2.05 ± 0.01 1.93 ± 0.03 1.79 ± 0.022.13 ± 0.01

Example 38 Solid State Properties of Powders Formulations VII and VIII

Formulations VII and VIII were also analyzed for amorphous/crystallinecontent and polymorphic form using high resolution X-ray powderdiffraction (XRPD). For XRPD, phase identification was performed toidentify any crystalline phases observed in each XRPD pattern. XRPDpatterns were collected using a PANalytical X'Pert Pro diffractometer(Almelo, The Netherlands). The specimen was analyzed using Cu radiationproduced using an Optix long fine-focus source. An elliptically gradedmultilayer mirror was used to focus the Cu Kα X-rays of the sourcethrough the specimen and onto the detector. The specimen was sandwichedbetween 3-micron thick films, analyzed in transmission geometry, androtated to optimize orientation statistics. A beam-stop was used tominimize the background generated by air scattering. Soller slits wereused for the incident and diffracted beams to minimize axial divergence.Diffraction patterns were collected using a scanning position-sensitivedetector (X′Celerator) located 240 mm from the specimen. Scans wereobtained over 3-60° with a step size of 0.017° and a step time of 70 s.As shown in FIG. 45A, peaks at approximately 6, 19, 24, 31 and 33°characteristic of leucine (leucine scan not shown) can be seen in thediffractogram for Formulation VII, indicating the presence ofcrystalline leucine in this powder (the peak at approximately 44° ineach scan is due to the sample holder). No crystallinity peakscharacteristic of either calcium lactate pentahydrate or sodium chloridewere observed in the diffractograms for either Formulations VIII andVII, indicating that these components were likely present in anamorphous form in these powders.

Modulated Differential Scanning calorimetry (mDSC) experiments wereperformed utilizing a DSCQ200 System from TA Instruments Inc.Approximately 10 mg of samples were placed inside hermetically sealedpans. mDSC conditions were: equilibration at 0° C. and modulation with aheating rate of 2° C./min, amplitude of 0.32° C. and period of 60 suntil 250° C. Glass transition temperatures were determined by theinflection point of the step change in the reversible heat flow versustemperature curve. Using this method, the glass transition temperature(T_(g)) of Formulation VIII was determined to be approximately 107° C.and that of Formulation VII approximately 91° C. (FIG. 45B).

Example 39 Effect of Calcium to Sodium Ion Molar Ratio in Dry PowderEfficacy in a Mouse Model of Bacterial Pneumonia

Dry powders with various molar ratios of calcium to sodium were alsotested for their ability to reduce bacterial infection in a mouse modelof pneumonia. In a whole-body exposure system (see Example 33), C57BL6mice were treated with either a leucine powder or dry powders having afixed calcium dose of 0.24 mg/kg, but various molar ratios of calcium tosodium: 1:0, 16:1, 8:1, 4:1, 2:1, 1:1 and 1:2. Two hours aftertreatment, mice were infected with Serotype 3 Streptococcus pneumoniaand, 24 hours after infection, euthanized and the bacterial burden oftheir lungs assessed as described previously (see Examples 26 and 34).The lung bacterial burden of mice in each group was determined and isshown as a percent of the bacterial burden in control mice. As shown inFIG. 46A, calcium and sodium-containing dry powders at allcalcium:sodium molar ratios (1:1-16:1) significantly reduced thebacterial burden in S. pneumonia infected mice.

Further, the ability of dry powders of the invention to treat micealready infected with bacteria was assessed. Thus, either leucine, or adry powder (0.31 Ca mg/kg) having a calcium to sodium molar ratio of 4:1(Formulation VIII) was administered to mice either 2 hours before S.pneumonia infection (prophylaxis, FIG. 46B) or 4 hours after S.pneumonia infection (treatment, FIG. 46B). Compared to leucine-treatedmice, Formulation VIII was able to not only reduce bacterial burden inmice when administered before bacterial infection, but was also able doso when given after mice were already infected by bacteria. Accordingly,the data indicated that calcium and sodium dry powders could be used totreat not only infections acquired after salt formulation treatment, butto also treat pre-existing and/or established bacterial and viralinfections.

Example 40 Effect of Leucine Load and Calcium:Sodium Molar Ion Ratio inTreating Ferret Influenza

Dry powders with calcium and sodium molar ion ratios of 1:2 (FormulationIII) and 8:1 (Formulation VI) were also tested for efficacy in reducingthe severity of influenza in a ferret flu model (see Example 28). In anose-only exposure system, ferrets (n=8) were exposed to a controlpowder of 100% leucine, Formulation III (10.0% leucine, 58.6% calciumlactate, 31.4% sodium chloride; 10.8% calcium ion) at 0.1 mg/kg, 0.3mg/kg or 0.9 mg/kg or to Formulation VI (39.4% leucine, 58.6% calciumlactate, 2.0% sodium chloride; 10.8% calcium ion) at 0.3 mg/kg. Theferrets were exposed to the powders 1 hour before infection, 4 hoursafter infection, then twice daily (BID). The body weights andsubcutaneous body temperatures of the animals were taken twice a daystarting at day 0, where the body temperatures taken 1 through 3 daysbefore the study were used as a baseline from which body temperaturechanges were calculated.

Ferrets treated with control leucine powders showed the typical increasein body temperatures at day 2 and day 5 post-influenza infection.Compared to control animals, however, both Formulation III andFormulation VI suppressed this increase in body temperature (FIG. 47A).Further, Formulation VI reduced the severe loss in body weight typicallyseen in influenza-infected ferrets, while Formulation III did so in adose-responsive manner. Thus, both powders were able to decrease theseverity of ferret influenza and could be used to treat viralinfections.

Example 41 Efficacy of Salt Formulations with Various Calcium:SodiumMolar Ion Ratios on Mouse Allergic Asthma

Dry powder formulations having various calcium and sodium ion molarratios but a fixed dose of calcium (0.24 mg/kg) were tested in the OVAmouse model of allergic asthma (Example 29). After sensitization toovalbumin, mice were treated with a leucine powder or dry powders at8:1, 4:1, 2:1, 1:1 or 1:2 Ca:Na molar ion ratios by whole-body exposure1 hour before and 4 hours after challenge of the sensitized mice withOVA on days 27 through 29 and twice on day 30. Bronchoaveolar lavageswere performed on day 31 and the total number of cells and eosinophilsdetermined by differential staining. The data depicted are the standarddeviation of 4-5 mice per group and representative of at least twodifferent studies. Dry powders with a higher ratio of calcium ion tosodium ion, that is those with Ca:Na molar ratio of 8:1, 4:1 and 2:1,had the greatest effect in reducing both total cell numbers (FIG. 48A)and eosinophils (FIG. 48B). These data suggested that the molar ratio ofcalcium to sodium ion could play a role in the broad anti-inflammatoryeffect of the dry powder formulations.

Example 42 Effect of Dry Powders with Different Molar Ratios of Calciumto Sodium Ion in TS Mouse-Associated Inflammation

To determine the efficacy of other calcium-sodium powders and of aonce-daily dosing regimen (QD), a similar study was performed using the4 day tobacco smoke (TS) mouse model described previously (see Example30). Formulation III (10.0% leucine, 58.6% calcium lactate, 31.4% sodiumchloride; 10.8% calcium ion; Ca:Na molar ratio 1:2) and Formulation VII(37.6% leucine, 58.6% calcium lactate, 4% sodium chloride; 10.8% calciumion; Ca:Na molar ratio 4:1) were tested in the COPD model. Two differentdoses of calcium were delivered using Formulation VII by increasing thenumber of capsules used. Doses were calculated as described previously(See Example 30). Six groups of mice were exposed to TS daily for 4days. Each group received one of the following treatments: FormulationIII, Formulation VII or a leucine control vehicle administered twicedaily (BID) 1 hour prior to and 6 hours after TS-exposure by whole-bodydry-powder inhalation. Formulation III was also administered on aonce-daily regimen (QD) 1 hour prior to TS-exposure and a just leucinecontrol powder administered 6 hours after TS-exposure. The p38 inhibitorADS 110836 was administered by the intra-nasal route (i.n.) 1 hour priorto TS-exposure. One further group (sham) was exposed to air instead ofTS for a similar period and received a leucine control powderadministered BID 1 hour prior to and 6 hours after air exposure. Animalswere euthanized by intra-peritoneal barbiturate anaesthetic overdose 24hours after the final exposure to either air (sham) or TS on day 5, anda bronchoalveolar lavage (BAL) was performed using 0.4 mL of phosphatebuffered saline (PBS). Cells recovered from the BAL were enumerated anddifferential cell counts carried out using cytospin prepared slides.

The leucine treated animals exposed to TS exhibited a 10-fold increasein total cell counts compared to air treated animals who were alsoadministered the control powder. In contrast, the p38 MAPK positivecontrol reference compound inhibited inflammation (FIG. 49A). As before,treatment twice daily (BID) with approximately 1.68 mg Ca ion/kg withFormulation III significantly reduced total cell counts to 45% of thatof the control animals. Treatment with the same dose of calcium onlyonce, 1 hour before TS exposure (QD) resulted in a similar reduction intotal cell counts (51%) (FIG. 49A). Formulation VII also reduced totalcell counts in the BAL fluid in a dose responsive manner compared to thecontrol group (45% reduction for the 0.68 mg Ca/kg dose and 58%reduction for the 1.41 mg Ca/kg dose). In addition, both Formulation IIIand Formulation VII significantly reduced the number of inflammatorycells, including macrophages (FIG. 49B), neutrophils (FIG. 49C) andlymphocytes (FIG. 49D), with the greatest effect occurring onmacrophages and neutrophil. In fact, Formulation VII reduced neutrophiland macrophage cell counts to a greater degree than the positive controlreference compound, the p38 MAPK inhibitor ADS110836. Surprisingly, thelower of the two doses of Formulation VII reduced inflammatory cellcounts to the same level as the high dose of Formulation III, despitethe delivery of approximately 3-times less calcium ion. Likewise, thehigh dose of Formulation VII exhibited the greatest reduction inneutrophils of all the treatments.

Collectively, the data suggested that calcium-sodium dry powders have asignificant impact in reducing airway inflammation and are suitabletherapies for treating/preventing inflammation, particularly thatassociated with respiratory diseases like asthma, COPD and CF. Further,the fact that the once-daily and twice-daily dosing treatments hadcomparable effects suggested that a once-daily dosing treatment regimencould be used therapeutically.

Example 43 Dry Powders Reduce the Expression of InflammatoryChemokines/Cytokines

In diseases like allergic asthma and COPD, the influx of inflammatorycells like eosinophils, macrophages and neutrophils into the airwaylumen in response to environmental insult is due to cellular release ofcytokines and/or chemokines. These cytokines/chemokines signal to inducethe chemotaxis of inflammatory cells to the airway lumen. Using thepreviously described tobacco smoke (TS) mouse model of COPD, studieswere undertaken to determine if the calcium-containing dry powders bothreduced inflammation and modulated inflammatory cytokine/chemokineexpression. Mice were exposed to TS for 4 consecutive days and treatedwith Formulation III or Formulation VII once daily 1 hour before TSexposure. Control animals were exposed to a dry powder formulation of100% leucine and a second control group was treated with leucine, butnot exposed to TS. At euthanasia, bronchoalveolar lavages (BAL) wereperformed and BAL samples were assayed for a panel of 13 differentcytokines and chemokines that have a role in the inflammation. Proteinlevels were assessed in a multiplex assay using Luminex technology andconcentrations of each protein were determined from standard curves.Data were analyzed by one-way ANOVA and the p values are shown beloweach group relative to the vehicle group * p<0.05. KC and MIP2 representtwo key neutrophil chemokines and perform a function analogous to IL-8in humans. KC and MIP2 expression was upregulated by exposure to TS (seeFIGS. 50A-B, Leu Air versus Leu bars). Treatment with either FormulationIII or VII reduced the BAL levels of KC (FIG. 50A) and MIP2 (FIG. 50B)compared to leucine treated animals. The data were similar to theeffects of these same formulations on neutrophil chemotaxis to the lungin the same animals and suggested that one mechanism by which the drypowder formulations reduced neutrophilic inflammation is through thereduction of chemokine levels that recruit these cells to the lung.These data further suggested that treatment with calcium-containingformulations modulates the biochemical and biological response of theairway epithelium and airway macrophages.

Example 44 Dry Powders Treat a Pathogen-Induced Acute Exacerbation ofMouse Allergic Asthma

Acute exacerbations in asthmatics and COPD patients are a significantcause of lung function decline, morbidity and mortality. Rhinovirusinfection is associated with a significant number of acute exacerbationsin both patient populations. Calcium-containing dry powder formulationsreduced rhinovirus infection in cultured epithelial cells (see Example13 and FIG. 13C). Preclinical models of rhinovirus in mice have beenhampered by the fact that major strains of rhinovirus do not bind tomouse ICAM-1 and therefore do not infect mouse cells. Recently, a mousemodel of rhinovirus infection using a minor strain (RV1B) has beendescribed (Bartlett N W et al. Nat Med. 2008 February; 14(2):199-204).Bartlett et al. describes both rhinovirus infection of naïve mice andrhinovirus infection of ovalbumin-challenged mice as a model of acuteexacerbations. Using these models, the efficacy of a calcium-sodium drypowder against rhinovirus infection and inflammation was evaluated. Therhinovirus exacerbation model is shown in FIG. 55 c.

BALB/c mice (n=5) were treated with different doses of Formulation VIIIBID for three days before intranasal infection with RV1B. On the day ofinfection, mice were treated 1 hour before and 4 hours after infection.Twenty-four hours after infection, lung inflammation was assessed bytotal and differential cell counts in bronchoalveolar lavage samples. Atthe lowest dose tested, Formulation VIII significantly reduced thenumber of total inflammatory cells and neutrophils compared to leucinecontrol treated animals (FIG. 51A). To extend these findings to anexacerbation like model, mice were sensitized to OVA by standardprotocol (see Example 29) and dosed BID on each day of OVA challenge.One hour after the final OVA challenge, mice were infected with RV1B.Twenty-four hours after infection, lung inflammation was assessed bytotal and differential cell counts in bronchoalveolar lavage samples.Rhinovirus infection was associated with increased neutrophilicinflammation compared to uninfected control animals (FIG. 51B).Formulation VIII reduced that neutrophilic inflammation compared toleucine control treated animals (one-way ANOVA; Tukey's multiplecomparison test) (FIG. 51B). Together, these data suggested that aninhaled calcium dry powder could reduce the frequency and severity ofacute exacerbations in patients with respiratory disease, in part, bydiminishing the inflammation associated with the infection.

Example 45 Calcium-Containing Dry Powders do not Cause AirwayHyperreactivity

In respiratory diseases and conditions, the inhalation of foreignparticles can often have adverse effects on the small airway of thelung. This can result in airway constriction leading to increased airwayresistance, work of breathing and, in extreme cases, a considerable riskto the health of a patient. Thus, it is vital that inhaled therapies,particularly in the setting of inflamed or hyper reactive airways, donot result in any unintended consequences such as bronchoconstriction.Accordingly, a study was undertaken to determine whether acalcium-sodium formulation (Formulation VIII) would have an adverseeffect on airway bronchoconstriction. Airway resistance was assessedutilizing dual chamber plethysmography. Briefly, mice were constrainedin a conical restrainer and placed in a device that consists of twosealed chambers; one encompassing the head and the other encompassingthe body with an airtight seal between the two. Pneumotachs measuredairflow in each individual chamber and specific airway resistance(sRaw), a direct measure of airway caliber, was calculated as a functionof the time delay between flow signals. In order to precisely determinethe influence of Formulation VIII on sRaw, 5 minutes of baseline sRawmeasurements were obtained and the mice were subsequently exposed to ahigh dose of Formulation VIII (0.90 mg Ca²⁺/kg). Exposure of the mice tothe dry powder was accomplished through the use of a whole body exposurechamber using a capsule-based dry powder inhaler system. Followingtreatment, 5 minutes of post-treatment sRaw measurements were obtained.Mice were then exposed to escalating doses of methacholine chloride(MCh) in 0.9% sodium chloride for inhalation via nebulization into thehead chamber for 10 seconds. The experimental procedure is shown in FIG.55D.

After each subsequent dose of MCh (0, 6.25, 12.5 25, and 50 mg/ml) thehead chamber was cleared and an additional 5 minutes of sRaw was taken.The average sRaw for each 5 minute period was calculated for each animaland normalized to baseline sRaw. This was repeated for two additionalgroups of mice, whereby the first group was treated with 100% leucinedry power in place of Formulation VIII, and the second group received asham treatment consisting of dry air only.

Surprisingly, treatment with Formulation VIII (and leucine) resulted inlittle change in sRaw and, instead, was statistically indistinguishablefrom the sham treatment (FIG. 52). In fact, when the animals wereexposed to nebulized saline for inhalation (0 mg/ml MCh), the magnitudeincrease in sRaw was higher than that which was seen during dry powdertreatment. In each group, sRaw increased with escalating MCh dose;however, at no point was there a significant difference in sRaw betweentreatment groups.

Overall, the data demonstrated that calcium dry powder treatment hadlittle influence on sRaw in healthy non-challenged airways and that acalcium dry powder does not adversely influence airway response duringperiods of bronchoconstriction. Unexpectedly, 0.9% sodium chloridesolution for inhalation, a widely utilized diluent for inhaled drugtherapies, resulted in a larger magnitude increase in sRaw than didFormulation VIII. These results clearly demonstrated thatcalcium-containing dry powders are not likely to inadvertently constrictsmall airways like some currently accepted therapies (e.g., mannitolinhalation therapy for cystic fibrosis) and could serve as a safe andeffective therapy for conditions like COPD, asthma and CF.

Example 46 In Vivo Sheep Mucociliary Clearance Studies Using FormulationVIII

A liquid and a dry powder formulation were evaluated in an establishedsheep mucociliary clearance (MCC) model. MCC was evaluated in fourhealthy sheep by measurement of the clearance of pulmonary Tc99m-labeledsulfur colloid aerosols that were delivered by inhalation. Immediatelyfollowing the treatment aerosol exposures, the radio-labeled sulfurcolloid aerosol was delivered to each of the sheep via the same aerosoldelivery system and MCC determined via the collection of serial images.

A Pari LC jet nebulizer operating with a single sheep exposure systemwas used to deliver Formulation 46-A (which is 9.4% CaCl₂ (w/v), 0.62%NaCl (w/v) in water, at a concentration resulting in a tonicity factorof 8 times isotonic). The nebulizer was connected to a dosimeter systemconsisting of a solenoid valve and a source of compressed air (20 psi).The output of the nebulizer is connected to a T-piece, with one endattached to a respirator (Harvard Apparatus Inc., Holliston, Mass.). Thesystem was activated for 1 second at the onset of the inspiratory cycleof the respirator, which was set at an inspiratory/expiratory ratio of1:1 and a rate of 20 breaths/minute. A tidal volume of 300 ml was usedto deliver the nebulized formulation. The nebulizer was filled with 4 mLof Formulation 46-A and run to dryness. A dry powder, Formulation VIII,was delivered with a similar exposure system but with a rotating brushgenerator (RBG1000, Palas) used to generate the dry powder aerosolinstead of the nebulizer. A 15 minute dose of the dry powder FormulationVIII was delivered with the aerosol continuously generated by the RBG.

The same aerosol exposure system as the liquid treatment was used todeliver aerosolized technetium labeled sulfur colloid (99mTC-SC)immediately after treatment. Animals were conscious, supported in amobile restraint, intubated with a cuffed endotracheal tube andmaintained conscious for the duration of the study.

After 99mTC-SC nebulization, the animals were immediately extubated andpositioned in their natural upright position underneath a gamma camera(Dyna Cam, Picker Corp., Nothford, Conn.) so that the field of image wasperpendicular to the animals' spinal cord. After acquisition of abaseline image, serial images were obtained at 5 min intervals for thefirst hour. All images were obtained and stored in the computer foranalysis. An area of interest was traced over the image corresponding tothe right lung of the animals, and counts were recorded. The left lungwas excluded from analysis because its corresponding image wassuperimposed over the stomach and counts could be affected by swallowedradiolabeled mucus. The counts were corrected for decay and clearanceexpressed as the percentage reduction of radioactivity present from thebaseline image.

The dose delivered for both formulations was measured in-vitro with abreathing simulator system drawing the inspiratory flow through filtersamples collected at the distal end of a tracheal tube. For theFormulation VIII dry powder, 10 filter samples of 1.5 minutes each wereassayed for deposited calcium by HPLC and the average rate of calciumdeposition was determined. From this the dose delivered in 15 minutes toa 50 kg sheep was calculated to be 0.5 mg Ca²⁺/kg. For the liquidFormulation 46-A, 1.5 minute filter samples were again assayed forcalcium content by HPLC and the dose delivered when running the 4 mLsolution to dryness was calculated for a 50 kg sheep to be 0.5 mgCa²⁺/kg. These measured doses correspond to the dose delivered from thedistal end of the tracheal tube to the sheep during treatment.

Each formulation was tested on 4 different sheep. The sheep mucociliaryclearance model is a well established model with vehicle clearancetypically measuring approximately 5-10% at 60 minutes after delivery ofthe radioactive aerosol (see for example Coote et al, 2009, JEPT329:769-774). It is known in the art that average clearance measurementsgreater than about 10% at 60 minutes post baseline indicate enhancedclearance in the model. Both the dry powder Formulation VIII and theliquid Formulation 46-A show enhanced mucociliary clearance in the sheepmodel, with average clearances±standard error at 60 minutes postbaseline of 16.7%±2.7% and 18.9%±1.2% of baseline radioactivityrespectively.

The mucociliary clearance was found to increase over the 60 minuteperiod post dosing. For example, the clearance at 10 minutes was2.9±2.3% of baseline and 4.5±1.4% of baseline, the clearance at 20minutes was 4.6±2.8% of baseline and 9.4±1.8% of baseline, the clearanceat 30 minutes was 7.7±4.0% of baseline and 10.6±1.7% of baseline, theclearance at 40 minutes was 12.1±2.5% of baseline and 13.6±0.1% ofbaseline, the clearance at 50 minutes was 13.1±2.6% of baseline and14.5±1.2% of baseline, the clearance at 60 minutes was 16.7±2.7% ofbaseline and 18.9±1.2% of baseline for Formulations VIII and 46-A,respectively.

The data presented herein show that calcium salt based dry powder andhypertonic liquid formulations can be used to increase mucociliaryclearance.

Example 47 In Vivo Canine Mucociliary Clearance Studies

The purpose of this study was to evaluate a liquid and a dry powderformulation in a canine mucociliary clearance (MCC) model. MCC wasevaluated in six healthy male Beagle dogs by serial image measurement ofthe removal of pulmonary Tc99m-labeled sulfur colloid aerosols that weredelivered by inhalation immediately following the treatment or controlaerosol exposures in a cross-over study.

A Pari LC Plus jet nebulizer operating with a single dog exposure systemutilizing a two-way valve and a dual phase respirator (HarvardApparatus; Holliston, Mass.) was used to deliver Formulation 47-A (whichis 9.4% CaCl2 (w/v), 0.62% NaCl (w/v) in water, at a concentrationresulting in a tonicity factor of 8 times isotonic). A drypowder—calcium based formulation (Formulation III) was delivered with asimilar exposure system but with a rotating brush generator (RBG1000,Palas) or with dry powder insufflators for the lowest dose. Untreatedand isotonic sodium chloride (0.9% w/v) were used as negative controls,while hypertonic saline (7% w/v) was used as a positive control in themodel. All doses were delivered for 15 minute durations with theexception of Formulation 47-A which was delivered for a duration of 7.5minutes and the low dose of Formulation III which was delivered by 4bolus deliveries from dry powder insufflators. Animals were anesthetizedwith propofol during the exposure and imaging periods and mechanicallyventilated during exposure. Immediately following the radiolabeledexposures, serial planar images were collected every two minutes until11 minutes and then every 5 minutes out to ˜33 minutes. Region ofinterest analysis was conducted on the lungs to determine the amount ofactivity remaining as a function of time and a fitted linear regressionparameter of the rate of radioactivity clearance calculated.

Treatment aerosols were collected from the end of the intubation tubewhile the Harvard pump was running prior to intubating the dogs todetermine the aerosol concentration (gravimetrically or chemically byHPLC) and size distribution (APS, TSI, Model 3321). To calculate theinhaled deposited dose, the respiratory minute volume (RMV) wascalculated allometrically (Bide et al. 2000, J. Appl. Toxicol.20:273-290). The estimated dose was then calculated using the followingformula: Dose=(C×RMV×T×DF)/BW, where C is the concentration of the testarticle in the exposure atmosphere, T is exposure time, BW is bodyweight and the deposition fraction (DF=30%) (Guyton AC. 1974, AmericanJournal of Physiology 150:70-77). The aerosol concentrations, calculateddelivered doses and resulting rate of MCC arc shown in Table 52.

TABLE 52 Aerosol treatment delivered doses and effect on MCC. AerosolDeposited Deposited Rate of Concentration Dose Dose radioactivity (mgdry (mg dry (mg Ca²/ clearance Treatment Group solids/L) solids/kg) kg)(%/minute) Untreated NA NA NA −0.233 Baseline Isotonic Saline 0.05 0.08NA −0.243 Hypertonic 0.4 0.58 NA −0.281 Saline Formulation 47-A 0.460.33 0.11 −0.285 Formulation III— 0.56 0.81 0.08 −0.275 low doseFormulation III— 1.9 2.75 0.3 −0.291 mid dose Formulation III— 3.5 5.060.55 −0.326 high dose

The high dose of Formulation III was found to have a slope of−0.326%/min which was significantly different from untreated baseline(p<0.05) over the 32 minute imaging interval. The mid dose ofFormulation III (slope=−0.291%/min) as well as Formulation 47-A(slope=−0.285%/min) treatment and the 7% hypertonic saline(slope=−0.281%/min) treatments were all found to be significantlydifferent from untreated baseline at a significance level of p<0.10,demonstrating an enhanced rate of mucociliary clearance in the dogscompared to untreated. The mid and high dose of Formualtion III enhancedclearance at least equivalent to 7% hypertonic saline. The 7.5 minutedose of Formulation 47-A provided an increase in mucociliary clearanceequivalent to the 15 minute dose of 7% hypertonic saline with only halfthe dosing duration at similar tonicities.

Example 48 Calcium-Containing Dry Powders Combined with Other ActiveAgents

A. Powder Preparation.

Feedstock solutions were prepared and used to manufacture dry powderscomprised of neat, dry particles containing calcium lactate, sodiumchloride, optionally leucine, and other pharmaceutically active agents.Table 53 lists the components of the feedstock formulations used inpreparation of the dry powders comprised of dry particles. Weightpercentages are given on a dry basis.

TABLE 53 Feedstock compositions of calcium-salt with otherpharmaceutically active agents Formulation Feedstock Composition (w/w) X75.0% calcium lactate, 5.0% sodium chloride, 18.96% leucine, 0.91%fluticasone propionate (FP), 0.13% salmeterol xinafoate (SX) XI 75.0%calcium lactate, 5.0% sodium chloride, 15.42% leucine, 4.0% fluticasonepropionate, 0.58% salmeterol xinafoate XII 75.0% calcium lactate, 5.0%sodium chloride, 15.31% leucine, 4.0% fluticasone propionate, 0.58%salmeterol xinafoate, 0.113% tiotropium bromide (TioB) XIII 75.0%calcium lactate, 5.0% sodium chloride, 18.85% leucine, 0.91% fluticasonepropionate, 0.13% salmeterol xinafoate, 0.113% tiotropium bromide XIV75.0% calcium lactate, 5.0% sodium chloride, 19.89% leucine, 0.113%tiotropium bromide XV 75.0% calcium lactate, 5.0% sodium chloride, 16.0%leucine, 4.0% fluticasone propionate XVI 75.0% calcium lactate, 5.0%sodium chloride, 15.89% leucine, 4.0% fluticasone propionate, 0.113%tiotropium bromide XVII 75.0% calcium lactate, 5.0% sodium chloride, 20%levofloxacin (Levo) XVIII 75.0% calcium lactate, 5.0% sodium chloride,17.5% leucine, 2.5% Immunoglobulin G (IgG) XIX 75.0% calcium lactate,5.0% sodium chloride, 19.9% leucine, 0.1% formoterol fumarate (FF) XX75.0% calcium lactate, 5.0% sodium chloride, 18.92% leucine, 1.08%albuterol sulfate (AS)

The feedstock solutions were made according to the parameters in Table54.

TABLE 54 Formulation Conditions Formulation: X XI XII XIII XIV XV Totalsolids (g) 4 5 4 4 3 4 Total volume water (L) 0.4 0.5 0.4 0.4 0.3 0.4Amount leucine in 1 L (g) 1.9 1.541 1.53 1.89 1.99 1.6 Amount FP in 1 L(g) 0.091 0.4 0.4 0 0 0.4 Amount SX in 1 L (g) 0.013 0.058 0.058 0 0 0Amount TioB in 1 L (g) 0 0 0.0113 0.0113 0.0113 0 Amount Levo in 1 L (g)0 0 0 0 0 0 Amount IgG in 1 L (g) 0 0 0 0 0 0 Amount FF in 1 L (g) 0 0 00 0 0 Amount AS in 1 L (g) 0 0 0 0 0 0 Formulation: XVI XVII XVIII XIXXX Total solids (g) 4 5 5 4 4 Total volume water (L) 0.4 0.5 0.5 0.4 0.4Amount leucine in 1 L (g) 1.59 0 1.75 1.99 1.892 Amount FP in 1 L (g)0.091 0 0 0 0 Amount SX in 1 L ( g) 0 0 0 0 0 Amount TioB in 1 L (g)0.0113 0 0 0 0 Amount Levo in 1 L (g) 0 2 0 0 0 Amount IgG in 1 L (g) 00 0.25 0 0 Amount FF in 1 L (g) 0 0 0 0.01 0 Amount AS in 1 L (g) 0 0 00 0.108 For all formulations, the liquid feedstock was batch mixed, thetotal solids concentration was 10 g/L, the amount of sodium chloride in1 liter was 0.5 g, and the amount of calcium lactate pentahydrate in 1liter was 10.6 g.

Formulation X through XX dry powders were produced by spray drying onthe Büchi B-290 Mini Spray Dryer (BÜCHI Labortechnik AG, Flawil,Switzerland) with powder collection on a 60 mL glass vessel from a HighPerformance cyclone. The system used the Büchi B-296 dehumidifier and anexternal LG dehumidifier (model 49007903, LG Electronics, EnglewoodCliffs, N.J.) was run constantly. Atomization of the liquid feedutilized a Büchi two-fluid nozzle with a 1.5 mm diameter. The two-fluidatomizing gas was set at 40 mm and the aspirator rate to 90%. Air wasused as the drying gas and the atomization gas. Table 55 below includesdetails about the spray drying conditions.

TABLE 55 Spray Drying Process Conditions Process Formulation ParametersX XI XII XIII XIV XV XVI XVII XVIII XIX XX Liquid 10 10 10 10 10 10 1010 10 10 10 feedstock g/L g/L g/L g/L g/L g/L g/L g/L g/L g/L g/L solidsconcentration (g/L) Process gas 180 180 180 180 180 180 180 179-180 100180 180 inlet temperature (° C.) Process gas 87-90 73-75 73-75 74-7584-93 76-79 76-80 91-95 55-57 80 74-78 outlet temperature (° C.) Processgas 667 667 667 667 667 667 667 667 667 667 667 flowrate (liter/hr, LPH)Atomization 35 28 28 28 28 28 28 35 32 28 28 gas flowrate (meters³/hr)Liquid 9.5 10 10 10 5.2 10 9.8 5.7 2.7 5.7 5.7 feedstock flowrate(mL/min)

B. Powder Characterization.

Powder physical and aerosol properties are summarized in Tables 57, 58,59 and 60 below. Values with ±indicates standard deviation of the valuereported. Table 56 shows that all formulations had an FPF_(TD)<3.4 μmgreater than 18%. Formulations X, XI, XIV, XV, XVI, XVII, XVIII and XIXeach had an FPF_(TD)<3.4 μm greater than 25%. Formulations X, XI, XV,and XVI each had FPF_(TD)<3.4 μm greater than 30%. All formulations hadan FPF_(TD)<5.6 μm greater than 40%. Formulations X, XI, XIV, XV, XVI,XVII, XVIII, and XIX had an FPF_(TD)<5.6 μm greater than 50%.Formulation XV had an FPF_(TD)<5.6 μm greater than 60%. All formulationshad a tapped density greater than 0.45 g/cc. Formulations X, XII, XIII,XIV, XV, XVII, XVIII, XIX and XX each had tapped densities greater than0.5 g/cc. Formulations X, XIII, XIV, XVII, XVIII, XIX and XX each hadtapped densities greater than 0.65 g/cc. All formulations had a HausnerRatio greater than 1.8. Formulations XII, XIV, XV, XVI, XVIII, and XIXeach had a Hausner Ratio greater than 2.0. Formulations XV, XVI, and XIXeach had a Hausner Ratio equal to or greater than 2.4.

TABLE 56 Aerodynamic and density properties ACI-2 Density FPF_(TD) < 3.4μm FPF_(TD) < 5.6 μm Bulk Tapped Form. % % g/cc g/cc H.R. X 30.48% ±0.66% 56.85% ± 0.17% 0.34 ± 0.01 0.66 ± 0.03 1.93 XI 30.77% ± 0.54%56.37% ± 0.24% N/A ± N/A N/A ± N/A N/A XII 18.64% ± 0.79% 45.30% ± 0.29%0.25 ± 0.09 0.51 ± 0.02 2.05 XIII 18.37% ± 0.65% 41.29% ± 1.14% 0.36 ±0.01 0.69 ± 0.01 1.93 XIV 28.25% ± 1.01% 53.19% ± 0.23% 0.36 ± 0.01 0.86± 0.03 2.38 XV 36.15% ± 0.55% 62.62% ± 1.83% 0.23 ± 0.02 0.58 ± 0.042.46 XVI 31.34% ± 0.37% 59.34% ± 0.21% 0.18 ± 0.01 0.48 ± 0.03 2.65 XVII25.16% ± 1.02% 52.17% ± 1.14% 0.34 ± 0.08 0.68 ± 0.02 1.98 XVIII 27.18%± 1.31% 52.38% ± 1.47% 0.36 ± 0.01 0.77 ± 0.02 2.15 XIX 27.84% ± 9.09%52.59% ± 8.34% 0.37 ± 0.00 0.90 ± 0.09 2.40 XX 23.78% ± 0.92% 47.71% ±0.60% 0.40 ± 0.07 0.79 ± 0.02 1.99 Form = Formulation; H.R. = HausnerRatio

Table 57 shows that all formulations had geometric diameters (Dv50) ofless than 3.5 μm at a dry powder inhaler flowrate of 60 LPM.Formulations X, XIII, XIV, XV, XVI, XVII, XVIII, XIX and XX had Dv50 ofless than 2.5 um at 60 LPM. All formulations had a Dv50 of less than 6.0μm at 15 LPM. Formulations X, XIII, XIV, XV, XVII, XVIII, XIX, and XXhad a Dv50 of less than 4.6 μm at 15 LPM. Formulations XIV, XV, XVII,XVIII, XIX and XX had a Dv50 of less than 4.0 μm at 15 LPM.

TABLE 57 Dispersibility properties (Spraytec geometric diameters)Dispersibility—Spraytec @ 60 LPM @ 15 LPM Formulation Dv50 (μm) GSD Dv50(μm) GSD X 2.10 ± 0.08 4.15 ± 0.45 4.38 ± 0.15 3.88 ± 0.24 XI 2.76 ±0.11 4.18 ± 0.50 4.93 ± 0.14 2.49 ± 0.50 XII 3.09 ± 0.32 4.68 ± 0.165.95 ± 0.31 3.39 ± 0.15 XIII 2.23 ± 0.11 4.15 ± 0.40 4.58 ± 0.12 4.19 ±0.18 XIV 1.92 ± 0.17 6.04 ± 0.42 2.51 ± 0.11 3.07 ± 0.40 XV 1.95 ± 0.065.47 ± 0.24 3.78 ± 0.08 3.25 ± 0.16 XVI 2.18 ± 0.08 5.24 ± 0.47 4.72 ±0.14 3.00 ± 0.19 XVII 2.01 ± 0.13 6.12 ± 0.45 2.83 ± 0.24 2.61 ± 0.42XVIII 1.80 ± 0.11 6.07 ± 0.22 2.23 ± 0.21 3.16 ± 0.55 XIX 2.11 ± 0.125.38 ± 0.67 2.60 ± 0.05 3.04 ± 0.19 XX 2.13 ± 0.08 5.83 ± 0.20 2.56 ±0.04 3.22 ± 0.20

Table 58 shows that all formulations had a capsule emitted particle mass(CEPM) of greater than 94% at 60 LPM. Formulations X, XI, XII, XIV, XV,XVI, XVII, XVIII, XIX and XX each had a CEPM of greater than 97% at 60LPM. All formulations had a CEPM of greater than 80% at 15 LPM, exceptXI. Formulations XII, XIV, XV, XVI, XVIII, XIX and XX each had a CEPM ofgreater than 90% at 15 LPM.

TABLE 58 Dispersitibilty properties (CEPM) Dispersibility—CEPM @ 60 LPM@ 15 LPM Formulation CEPM CEPM X 97.48% ± 0.49% 80.33% ±  4.27% XI99.09% ± 0.24% 59.92% ± 27.96% XII 97.19% ± 0.25% 93.15% ±  3.90% XIII94.80% ± 1.53% 82.46% ±  4.61% XIV 97.83% ± 0.45% 95.99% ±  0.32% XV98.05% ± 0.39% 92.22% ±  3.48% XVI 103.32% ± 2.01% 101.23% ±  2.07% XVII99.57% ± 0.00% 80.41% ±  0.32% XVIII 99.71% ± 0.16% 98.08% ±  0.57% XIX100.22% ± 0.22% 98.06% ±  0.47% XX 99.87% ± 0.22% 98.10% ±  0.21%

Table 59 shows that all measured formulations had a Dv50 using the RODOSat its 1.0 bar setting of less than 2.5 μm. Formulations X, XIII, XIV,XV, XVI, XVII, and XVIII each had a Dv50 of less than 2.2 μm.Formulations X, XIII, XV, XVI, and XVII each had a Dv50 of less than 2.0μm. All measured formulations had a RODOS Ratio for 0.5/4 bar of lessthan 1.2. All measured formulations had a RODOS Ratio for 1/4 bar ofless than 1.1.

TABLE 59 Dispersitibilty properties (Geometric diameter using RODOS)RODOS 0.5 bar 1.0 bar 4.0 bar 0.5/4 1/4 Formulation Dv50 (μm) GSD Dv50(μm) GSD Dv50 (μm) GSD bar bar X 1.92 2.15 1.78 2.12 1.67 2.04 1.15 1.07XI N/A N/A N/A N/A N/A N/A N/A N/A XII 2.64 2.21 2.40 2.15 2.24 2.171.18 1.07 XIII 1.87 2.12 1.95 2.17 2.36 2.13 0.79 0.83 XIV 2.01 2.162.12 2.22 1.99 2.19 1.01 1.07 XV 2.12 2.16 1.84 2.15 1.92 2.16 1.10 0.96XVI 2.13 2.15 1.83 2.14 1.87 2.18 1.14 0.98 XVII 1.93 2.23 1.83 2.241.69 2.17 1.14 1.08 XVIII 2.08 2.12 2.03 2.09 1.95 2.15 1.07 1.04 XIX2.13 2.14 2.26 2.20 2.15 2.25 0.99 1.05 XX 2.24 2.14 2.22 2.19 2.23 2.221.00 1.00

C. Anti-Inflammatory Efficacy of a Co-Formulation of a Calcium Salt withFluticasone Propionate and Salmeterol Xinafoate (Formulation XI) in anOVA Mouse Model of Allergic Asthma

Formulation XI was evaluated in a mouse model of allergic asthma usingovalbumin (OVA) as an allergen. The model has been described and shownpictorally in Example 29.

In this model, mice were sensitized to OVA over a period of two weeksand subsequently challenged, via a liquid aerosol, with OVA (Example29). This challenge induced lung inflammation and increased airwayhyperreactivity in response to an airway challenge. The principle changein inflammation was an increase in the number of eosinophils in thelungs. Similar changes in lung inflammation and pulmonary function havebeen observed in humans with asthma.

Balb/c mice were sensitized and challenged to OVA, as per thesensitization protocol described in Example 29. Mice were treated withPlacebo-B dry powder (98% leucine, 2% NaCl, w/w on a dry basis),Formulation 48-A (30% leucine, 65.4% NaCl, 4.0% fluticasone propionateand 0.13% salmeterol xinafoate, w/w on a dry basis), and Formulation XI(75.0% calcium lactate, 15.31% leucine, 5.0% NaCl, 4.0% fluticasonepropionate and 0.58% salmeterol xinafoate, w/w on a dry basis).Treatments were made in a whole body exposure chamber using a capsulebased dry powder inhaler system. On the final day of the study (day 31),mice were euthanized and bronchoalveolar lavages (BAL) were performed.The total number of cells per BAL was determined. In addition, thepercentage and total number of eosinophils, neutrophils, macrophages,and lymphocytes were determined by differential staining.

The effect of Formulation XI on inflammation was assessed. Based on theliterature, such as, (Ohta, S. et al. (2010), “Effect of tiotropiumbromide on airway inflammation and remodeling in a mouse model ofasthma”, Clinical and Experimental Allergy 40:1266-1275), and(Riesenfeld, E. P. (2010), “Inhaled salmeterol and/or fluticasone altersstructure/function in a murine model of allergic airways disease”,Respiratory Research, 11:22), fluticasone propionate (FP) is known toreduce eosinophilic cells and total cellularity in the mouse OVA model.

What was unknown in the art was the effect of co-formulating FP with acalcium salt formulation. Therefore, Formulation XI was tested. Theresults in Table 60 show that for a similar dose (mg FP/kg mouse bodyweight), Formulation XI was equally as efficacious in reducingeosinophilic cells and total cellularity as when the FP was formulatedwithout the calcium salt (Formulation 48-A).

TABLE 60 Formulation XI reduces eosinophilic and total cellularinflammation in a murine model of allergic asthma Placebo-B Formulation48-A Formulation XI cells * 10⁶/ml Std Dev cells * 10⁶/ml Std Devcells * 10⁶/ml Std Dev Eosinophils 0.55 0.27 0.11 0.10 0.11 0.09 Totalcells 1.38 0.50 0.49 0.20 0.71 0.91 (Cellularity)

D. Effect of Co-Formulations of a Calcium Salt and Salmeterol Xinafoateand Tiotropium Bromide (Formulations XI and XVII, Respectively) onSpecific Airway Resistance in a Mouse OVA Model

The sensitization of mice with OVA and subsequent challenging of micewith OVA was achieved, as described in Example 29. In addition tochanges in inflammation, mice sensitized and challenged with OVA exhibitincreased airway hyperreactivity, which can be measured as changes inairway resistance following bronchoprovocation. Pulmonary functiontesting was conducted one hour following treatment on day 30. Thisinvolved measuring the specific airway resistance (sRaw) in the mice.Baseline sRaw measurements were taken for 5 minutes. The micesubsequently underwent a methacholine (MCh) challenge for assessingpulmonary function with escalating concentrations of MCh delivered vianebulization in a head chamber using doses of MCh of 0 mg/ml, 50 ing/mlor 100 mg/ml.

The mice were challenged to test their pulmonary function according tothe methods described in Example 45. It was known from the literature,for example, (Schutz, N. (2004), “Prevention of bronchoconstriction insensitized guinea pigs: efficacy of common prophylactic drugs”, RespirPhysiol Neurobiol 141(2): 167-178), and (Ohta, S. et al. (2010), “Effectof tiotropium bromide on airway inflammation and remodeling in a mousemodel of asthma”, Clinical and Experimental Allergy 40:1266-1275), thatboth salmeterol xinafoate (SX) and tiotropium bromide (TioB) enhancedpulmonary function, resulting in lower sRaw values, for animals andhuman beings challenged with methacholine chloride (MCh) in 0.9% sodiumchloride for inhalation.

While the effects of SX and TioB on sRaw were known from the literature,the effect of co-formulating SX and TioB formulations with a calciumsalt were unknown. Formulations XI (75.0% calcium lactate, 15.31%leucine, 5.0% NaCl, 4.0% fluticasone propionate and 0.58% salmeterolxinafoate, w/w on a dry basis), XIV (75.0% calcium lactate, 19.89%leucine, 5.0% NaCl, and 0.113% tiotropium bromide, w/w on a dry basis),48-A (30% leucine, 65.4% NaCl 4.0% fluticasone propionate and 0.13%salmeterol xinafoate, w/w on a dry basis), and 48-B (34.47% leucine,65.42% NaCl and 0.113% tiotropium bromide, w/w on a dry basis) weretested. Non-calcium containing Formulations 48-A and 48-B were tested inorder to contrast the efficacies of the calcium-containing FormulationsXI and XIV, respectively. Results from pulmonary function testing areshown in FIG. 53 and FIG. 54 for Formulations XI and XIV, respectively.These data show that calcium-containing Formulation XIV matched thepositive control, Formulation 48-B, and completely eliminates airwayhyperreactivity in response to methacholine challenge in an OVA model ofallergic asthma. Treatment with Formulation XI did not match thereduction in sRaw that Formulation 48-A achieved, however, thevariability within the group treated with Formulation XI overlapped thatof Formulation 48-A and the mean reduction was lower than that observedwith Placebo-B.

E. Efficacy of Co-Formulations of a Calcium Salt with FluticasonePropionate and Salmeterol Xinafoate in an LPS Mouse Model of Acute LungInjury

In this study, a mouse model of acute lung injury was used to study theeffects of calcium and sodium formulations combined with othertherapeutics on pulmonary inflammation. Mice were exposed to aerosolizedlipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa. Thischallenge resulted in lung inflammation and caused changes in pulmonaryfunction. The principle change in inflammation was an increase in thenumber of neutrophils in the lungs. Similar changes in lung inflammationand pulmonary function were observed in humans suffering from acute lunginjury.

Mice were exposed to whole body exposure with nebulized LPS, 1.12 mg/ml,for 30 minutes. Treatment with dry powder Formulations XI (75.0% calciumlactate, 15.31% leucine, 5.0% NaCl, 4.0% fluticasone propionate and0.58% salmeterol xinafoate, w/w on a dry basis) was performed 1 hourfollowing LPS exposure using a whole body exposure chamber using acapsule based dry powder inhaler system. Animals were treated with 2, 90mg capsules corresponding to approximately 0.32 mg Ca²⁺/kg delivered tothe lung. To compare the influence of formulations with and withoutcalcium salt, an additional group of animals was exposed to anequivalent amount (i.e. mg of fluticasone/kg of body mass) of anadditional powder consisting of Formulation 48-A (30% leucine, 65.4%NaCl, 4.0% fluticasone propionate and 0.13% salmeterol xinafoate). Aseparate group of animals was treated with 2, 30 mg capsules ofPlacebo-B control powder (98% leucine, 2% NaCl). Three hours followingdry powder treatment, all mice were euthanized and underwent whole lunglavage for determination of total and differential cell counts.

As shown in Table 61, treatment of mice with Formulation XIsignificantly reduced total cell counts and neutrophils in the BAL fluidwhen compared with animals exposed to Placebo-B and reduced inflammatorycells to a greater extent than the calcium-free Formulation 48-A. Thus,treatment of mice with Formulation XI significantly reduced lunginflammation in an LPS model of acute lung injury.

TABLE 61 Formulation XI reduces inflammation in a rodent model of acutelung injury. Placebo-B Formulation 48-A Formulation XI Std Std Stdcells * 10⁶/ml Dev cells * 10⁶/ml Dev cells * 10⁶/ml Dev Neutrophils1.80 0.69 1.27 0.47 1.01 0.46 Total cells 1.94 0.71 1.37 0.52 1.12 0.47(Cellularity)

F. Anti-Bacterial Efficacy of Co-Formulations of a Calcium Salt andLevofloxacin on in a Pseudomonas aeruginosa Mouse Model

A mouse model of bacterial infection was used to evaluate the efficacyof Formulation XVII in vivo. Neutropenia was induced by injection ofcyclophosphamide (100 mg/Kg) on days −4 and −1. Bacteria (Pseudomonasaeruginosa) were grown overnight in 2 ml of Luria Bertani broth at 37°C. and approximately 5000 CFU were delivered per mouse via intranasaladministration in 50 μl of PBS. Four hours following infection theanimals were treated with Placebo-B powder (98% leucine, 2% NaCl),Formulation 48-C (27% leucine, 52% NaCl and 20% levofloxacin), andFormulation XVII (75.0% calcium lactate, 5.0% NaCl, 20% levofloxacin)using a whole body exposure chamber using a capsule based dry powderinhaler system. The next day, animals were euthanized and the lungs andthe spleen were harvested and homogenized to determine lung bacterialload and systemic bacterial load, respectively. Homogenates wereserially diluted on tryptin-soyagar plates and allowed to incubateovernight at 37° C. The following day, colony forming units were countedand CFU/ml for each the lung and the spleen was calculated.

The results are shown in Table 62. It was seen that Formulations XVIIand 48-C significantly reduced bacterial burden in the lung by more than5 log₁₀ CFU and in the spleen by almost 100-fold compared to thePlacebo-B treated animals. Thus, treatment of mice with Formulation XVIIsignificantly reduced lung and systemic bacterial burden duringPseudomonas aeruginosa infection. It was observed from these data thatthe presence of calcium in levofloxacin dry powder formulations did nothave a deleterious effect on the efficacy of levofloxacin. This is asurprising result given the literature which says that magnesium andcalcium based antacids deleteriously affect the bioavailability oflevofloxacin taken through the gastrointestinal tract. (Flor, S. et al.(1990), “Effects of Magnesium-Aluminum Hydroxide and Calcium CarbonateAntacids on Bioavailability of Ofloxacin”, Antimicrobial Agents andChemotherapy 34(12): 2436-2438), and (Pai, M P. et al. (2006), “Alteredsteady state pharmacokinteics of levofloxacin in adult cystic fibrosispatients receiving calcium carbonate”, J. Cyst. Fibros., August;5(3):153-7). (Ofloxacin is a racemic mixture, which consists of 50%levofloxacin, which is known to be biologically active, and 50% of itsenantiomer.)

TABLE 62 Formulation XII reduces bacterial burden during Pseudomonasaeruginosa infection Placebo Formulation 48-C Formulation XVII Std StdStd CFU/ml Dev CFU/ml Dev CFU/ml Dev Lung 2.85 × 10⁸ 2.88 × 10⁸ 2.08 ×10⁴ 3.87 × 10⁴ 9.22 × 10³ 1.78 × 10³ Spleen 1.57 × 10⁵ 1.78 × 10⁵ 2.16 ×10³ 6.81 × 10² 2.53 × 10³ 2.41 × 10³

G. Co-Formulation of a Calcium Salt and a Protein (Formulation XVIII)Provides for Delivery of the Protein Both Locally in the Lungs andSystemically

In this study, Formulation XVIII (75.0% calcium lactate, 17.5% leucine,5.0% sodium chloride, 2.5% bovine immunoglobulin G (IgG), w/w on a drybasis) was used to determine if calcium containing dry powderformulations can be used to deliver proteins to the lung and if this drypowder can be used to deliver proteins systemically.

In this study, mice were treated with Formulation XVIII using a wholebody exposure chamber using a capsule based dry powder inhaler system.Animals were then treated with 2, 4 or 6 capsules of Formulation XVIIIwith another group of animals were treated with 6 capsules of Placebo-Bcontrol powder (98% leucine, 2% NaCl). The placebo controls were run toensure that there was no cross reactivity with the bovine IgG assay andnative mouse proteins in either the serum or the broncho-alveolar lavage(BAL). Immediately following DP treatment the animals were euthanized,underwent BAL and serum was collected. lavage fluid and serum were thenassayed for bovine IgG using a commercially available ELISA kit. Theresults are shown in Table 63. Placebo-B (n=3 animals, data not reportedin table) was below the detectable range of the assay, which wasindicative that there was no cross reactivity between the bovine IgG andthe native mouse protein in either the serum or the BAL. It can be seenthat IgG delivered to the lung increases stepwise with increasing numberof capsules delivered to the animals. Furthermore, while treatment with2 or 4 capsules of Formulation XVIII resulted in slight increases inserum IgG content that were in the range of the detection limit of theELISA kit, treatment with 6 capsules IgG resulted in an increase toapproximately 100 ng/ml IgG. Assuming an approximate serum volume of 2ml, this would suggest that, on average, 200 ng of IgG was deliveredsystemically with 6 capsules of Formulation XVII treatment. Thisdemonstrated that calcium-containing dry powders can be utilized todeliver proteins systemically.

TABLE 63 Calcium containing, inhaled dry powders can be utilized todeliver proteins to the lungs and systemically Lung IgG Serum IgG IgG(ng) Std Dev IgG (ng/ml) Std Dev Form. XVIII 100.61 39.45 3.68 6.05 (2capsules) Form. XVIII 148.32 28.90 6.63 10.58 (4 capsules) Form. XVIII274.73 72.52 107.41 49.41 (6 capsules) n = 6 animals each for the 2, 4,and 6 capsule groups

Example 49 Magnesium-Containing Dry Powders Combined with ActivePharmaceutical Agents

A. Powder Preparation.

Feedstock solutions were prepared in order to manufacture dry powderscomprised of neat, dry particles containing a magnesium salt, optionallya non-salt excipient, and at least one pharmaceutical active agent.Table 64 lists the components of the feedstock formulations used inpreparation of the dry powders comprised of dry particles. Weightpercentages are given on a dry basis.

TABLE 64 Feedstock compositions of calcium-salt with otherpharmaceutically active agents Formulation Feedstock Composition (w/w)XXI 89.89% magnesium lactate, 10.0% maltodextrin, 0.113% tiotropiumbromide (TioB) XXII 65.42% magnesium sulfate, 34.47% leucine, 0.113%tiotropium bromide (TioB) XXIII 65.42% magnesium sulfate, 34.0% leucine,0.58% salmeterol xinafoate XXIV 65.42% magnesium sulfate, 29.89%leucine, 4.0% fluticasone propionate, 0.58% salmeterol xinafoate, 0.113%tiotropium bromide

The feedstocks solutions were made according to the conditions in Table65.

TABLE 65 Formulation Conditions Formulation: XXI XXII XXIII XXIV Totalsolids (g) 2 3 3 4 Total volume water (L) 0.25 0.3 0.3 1.45 Amountmagnesium 7.19 6.54 6.54 2.35 lactate (ML) or magnesium sulfate (MS) in1 L (g) Amount of maltodextrin 0.8 3.45 3.4 0.275 (Malto) or leueine(Leu) (g) Amount FP in 1 L (g) 0 0 0 0.11 Amount SX in 1 L (g) 0 0 0.0580.016 Amount TioB in 1 L (g) 0.009 0.0113 0 0.0036

Formulation XXI through XXIV dry powders were produced by spray dryingon the Büchi B-290 Mini Spray Dryer (BÜCHI Labortechnik AG, Flawil,Switzerland) with powder collection on a 60 mL glass vessel from a HighPerformance cyclone. The system used the Büchi B-296 dehumidifier and anexternal LG dehumidifier (model 49007903, LG Electronics, EnglewoodCliffs, N.J.) was run constantly. Atomization of the liquid feedutilized a Büchi two-fluid nozzle with a 1.5 mm diameter. The two-fluidatomizing gas was set at 40 mm and the aspirator rate to 90%. Air wasused as the drying gas and the atomization gas. Table 66 below includesdetails about the spray drying conditions.

TABLE 66 Spray Drying Process Conditions Formulation Process ParametersXXI XXII XIII XXIV Liquid feedstock solids 8 10 10 2.75 concentration(g/L) Process gas inlet temperature 115 115 115 180 (° C.) Process gasoutlet temperature 63 65-67 65 68-73 (° C.) Process gas flowrate(liter/hr, 667 667 667 667 LPH) LPH LPH LPH LPH Atomization gas flowrate35 m³/h 35 m³/h 35 m³/h 35 m³/h (meters³/hr) Liquid feedstock flowrate2.5 2.5 2.5 12.1 (mL/min)

B. Powder Characterization.

Powder physical and aerosol properties are summarized in Tables 68, 69,and 70 below. Values with ±indicates standard deviation of the valuereported. Table 67 shows that all formulations had an FPF_(TD)<3.4 μmgreater than 25%. Formulations XXI, XXII, and XXIII each had anFPF_(TD)<3.4 μm greater than 35%. Formulations XXII and XXIII each hadFPF_(TD)<3.4 μm greater than 39%. All formulations had an FPF_(TD)<5.6μm greater than 50%. Formulations XXI, XXII, and XXIII had anFPF_(TD)<5.6 μm greater than 60%. Formulation XXIII had an FPF_(TD)<5.6μm greater than 68%. All formulations had a tapped density greater than0.70 g/cc. Formulations XXII and XXIII each had tapped densities greaterthan 0.90 g/cc. All formulations had a Hausner Ratio greater than 1.7.Formulations XXII and XXIII had Hausner Ratios greater than 2.0.

TABLE 67 Aerodynamic and density properties ACI-2 Density FPF_(TD) < 3.4μm FPF_(TD) < 5.6 μm Bulk Tapped Form. % % g/cc g/cc H.R. XXI 35.50% ±1.22% 63.47% ± 0.33% 0.40 ± 0.01 0.71 ± 0.00 1.77 XXII 39.87% ± 0.71%61.27% ± 2.10% 0.53 ± 0.02 1.09 ± 0.04 2.08 XXIII 47.74% ± 1.48% 68.41%± 1.02% 0.39 ± 0.01 0.94 ± 0.07 2.44 XXIV 27.73% ± 2.59% 51.51% ± 0.74%0.40 ± 0.00 0.72 ± 0.02 1.77 Form. = Formulation; H.R. = Hausner Ratio

Table 68 shows that all formulations had geometric diameters (Dv50) ofless than 2.2 um at a dry powder inhaler flowrate of 60 LPM.Formulations XXI, XXII, and XXIII had Dv50 of less than 2.0 um at 60LPM. Formulations XXI, XXII, and XXIII had a Dv50 of less than 2.5 um at15 LPM.

TABLE 68 Geometric Diameters Dispersibility—Spraytec @ 60 LPM @ 15 LPMFormulation Dv50 (μm) GSD Dv50 (μm) GSD XXI 1.77 ± 0.12 5.96 ± 0.11 2.37± 0.09 2.74 ± 0.37 XXII 1.99 ± 0.13 6.40 ± 0.54 2.27 ± 0.08 3.16 ± 0.26XXIII 1.32 ± 0.08 6.72 ± 0.40 2.23 ± 0.09 3.26 ± 0.21 XXIV 2.12 ± 0.074.92 ± 0.36 6.21 ± 0.60 4.94 ± 0.32

Table 69 shows that all formulations had a capsule emitted particle mass(CEPM) of greater than 97% at 60 LPM. All formulations had a CEPM ofgreater than 80% at 15 LPM. Formulations XXI, XXII, and XXIII each had aCEPM of greater than 92% at 15 LPM. Formulations XXII and XXIII each hada CEPM of greater than 97% at 15 LPM.

TABLE 69 Dispersitibilty properties Dispersibility—CEPM @ 60 LPM @ 15LPM Formulation CEPM CEPM XXI 97.06% ± 0.40% 92.57% ± 1.36% XXII 99.43%± 0.33% 97.14% ± 0.35% XXIII 98.56% ± 0.23% 97.32% ± 0.99% XXIV 98.41% ±0.28% 80.79% ± 16.16%

Table 70 shows that all formulations had a Dv50 using the RODOS at its1.0 bar setting of less than 2.2 μm. Formulations XXI, XXIII, and XXIVeach had a Dv50 of less than 1.9 μm. Formulation XXIV had a Dv50 of 1.57μm. All measured formulations had a RODOS Ratio for 0.5/4 bar of lessthan 1.2. All measured formulations had a RODOS Ratio for 1/4 bar ofless than 1.1.

TABLE 70 Dispersitibilty properties (Geometric diameter using RODOS)RODOS 0.5 bar 1.0 bar 4.0 bar 0.5/4 1/4 Formulation Dv50 (μm) GSD Dv50(μm) GSD Dv50 (μm) GSD bar bar XXI 1.93 2.14 1.83 2.11 1.90 2.16 1.020.96 XXII 2.06 2.25 2.19 2.17 2.09 2.23 0.99 1.05 XXIII 1.90 2.12 1.852.16 1.78 2.13 1.07 1.04 XXIV 1.76 2.08 1.57 2.03 1.53 2.01 1.15 1.03

Example 50 Effect of a Magnesium-Containing Formulation on AirwayEosinophilic Inflammation and Pulmonary Function in a Model of AllergicInflammation Asthma

A. Inflammation

Dry powder Formulation XXV comprising leucine (37.5%), magnesium lactate(58.3%) and sodium chloride (4.2%) was evaluated in a mouse model ofallergic asthma using ovalbumin (OVA) as an allergen. The model has beendescribed and the dosing protocol shown pictorially in Example 29.

In this model, Balb/c mice were sensitized to OVA over a period of twoweeks and subsequently challenged, via a liquid aerosol of OVA (graphicin Example 29). This challenge induced lung inflammation and increasedairway hyperreactivity in response to an airway challenge. The principlechange in inflammation was an increase in the number of eosinophils inthe lungs. Similar changes in lung inflammation and pulmonary functionhave been observed in humans with asthma.

Mice were treated with Placebo-B dry powder (98% leucine, 2% NaCl, w/won a dry basis) or Formulation XXV. Treatments were administered by awhole body exposure chamber using a capsule based dry powder inhalersystem. On the final day of the study (day 31), mice were euthanized andbronchoalveolar lavages (BAL) were performed. The total number of cellsper BAL was determined. In addition, the percentage and total number ofeosinophils, neutrophils, macrophages, and lymphocytes were determinedby differential staining.

The effect of Formulation XXV on inflammation was assessed. Based on theliterature, magnesium was not known to reduce eosinophilic cells andtotal cellularity in the mouse OVA model. This knowledge was confirmedin this experiment. The eosinophilic and total cell count was notsignificantly different between Formulation XXV and Placebo-B, asreported in Table 71.

TABLE 71 Formulation XXV does not reduces eosinophilic and totalcellular inflammation in a murine model of allergic asthma Placebo-BFormulation XXV cells*10⁶/ml Std Dev cells*10⁶/ml Std Dev Eosinophils0.35 0.20 0.31 0.10 Total cells 1.55 0.53 1.31 0.26 (Cellutarity)

B. Pulmonary Function

The sensitization of mice with OVA and subsequent challenging of micewith OVA was described above. Beside acquiring elevated eosinophilicinflammation, these OVA sensitized mice also developed increased airwayhyperreactivity, which can be measured as changes in airway resistancefollowing bronchoprovocation. Based on the literature (Okayama, H. etal. (1987), “Bronchodilating effect of intravenous magnesium sulfate inbronchial asthma”, JAMA, February 27; 257(8):1076-8), magnesium sulfateis known to reduce bronchoconstriction following intravenousadministration. What was unknown is whether inhaled delivery ofmagnesium salts to the airway would have a similar impact onbronchoconstriction in a preclinical model. To test the efficacy ofFormulation XXV in reducing the mice's susesptibility to airwayhyperreactivity, pulmonary function testing was conducted one hourfollowing treatment on day 30. This involved measuring the specificairway resistance (sRaw) in the mice. Baseline sRaw measurements weretaken for 5 minutes. The mice subsequently underwent a methacholine(MCh) challenge for assessing pulmonary function with escalatingconcentrations of MCh delivered via nebulization in a head chamber usingdoses of MCh of 0 mg/ml, 25 mg/ml or 50 mg/ml.

The mice were challenged to test their pulmonary function according tothe methods described in Example 45. From a survey of the literature, itwas possible that Formulation XXV would have efficacy in reducing airhyperreactivity, and thereby result in lower sRaw values, for animalsand human beings challenged with methacholine chloride (MCh) in 0.9%sodium chloride for inhalation.

Results from pulmonary function testing are shown in Table 72 forFormulations XXV and Placebo-B. These data show thatmagnesium-containing Formulation XXV matched the placebo, a negativecontrol, and that Formulation XXV did not mimic the literature resultwhere intravenous administration of magnesium sulfate reducedbronchoconstriction.

TABLE 72 Magnesium-containing Formulation XXV was not observed to have asignificant effect on pulmonary function as measured in the MChchallenge test. Specific Airway Resistance Placebo-B Formulation XXVcmH₂O*s Std Dev cmH₂O*s Std Dev Baseline 3.70 0.82 3.33 1.31 PBS 4.972.54 3.90 0.60 25 mg/ml MCh 21.36 15.21 22.97 6.54 50 mg/ml MCh 25.5311.91 24.61 8.08

Formulation XXV was tested in a mouse model of allergic inflammation,the OVA model. Formulation XXV was found not to cause a significantdecrease in eosinophilic or total inflammation cell counts vs.Placebo-B. Likewise, Formulation XXV was tested to ascertain its role inpulmonary function. Formulation XXV was found not to cause a significantdecrease in sensitivity to MCh challenge vs. Placebo-B.

Example 51 Effect of Dry Powders with Magnesium in TS Mouse-AssociatedInflammation

To determine the efficacy of a magnesium formulation in a COPD-likemodel of lung inflammation, a study was performed using the 4 daytobacco smoke (TS) mouse model. This model has been previously describedin Example 30. Formulation XXVI (19.6% leucine, 75.0% magnesium lactate,5.4% sodium chloride) and Formulation VIII (20.0% leucine, 75.0% calciumlactate, 5.0% sodium chloride) were tested in the COPD-like model. Thedoses of calcium and magnesium administered to the mice were matched ona μmol of salt/kg basis and doses were achieved by the delivery ofFormulations XXVI and VIII in 6 capsules of each formulation in theexperiment. Doses were calculated as described previously (See Example30). Six groups of mice were exposed to TS daily for 4 days. Each groupreceived one of the following treatments: Formulation XXVI, FormulationVIII or a leucine control vehicle administered once daily (QD) 1 hourprior to TS-exposure by whole-body dry-powder inhalation. The p38inhibitor ADS 110836 was administered by the intra-nasal route (i.n.) 1hour prior to TS-exposure. One further group (sham) was exposed to airinstead of TS for a similar period and received a leucine control powderadministered BID 1 hour prior to air exposure. Animals were euthanizedby intra-peritoneal barbiturate anaesthetic overdose 24 hours after thefinal exposure to either air (sham) or TS on day 5, and abronchoalveolar lavage (BAL) was performed using 0.4 ml, of phosphatebuffered saline (PBS). Cells recovered from the BAL, were enumerated anddifferential cell counts carried out using cytospin prepared slides.

The leucine treated animals exposed to TS exhibited an 8.4-fold increasein total cell counts compared to air treated animals who were alsoadministered the control powder. As before, QD treatment withapproximately 1.68 mg Ca ion/kg with Formulation VIII significantlyreduced total cell counts to 53% of that of the control animals.Treatment with the same dose of Mg ion/kg did not result in astatistically significant reduction in total cell counts (Table 73). Asimilar result was seen in in the inflammatory cell counts formacrophages (Table 73), neutrophils (Table 73) and lymphocytes (Table73), i.e. Formulation VIII reduced the cell counts for each category bya statistically significant amount. In contrast, Formulation XXVI didnot reduce the total cell counts, neutrophils or lymphocytes tostatistically significant levels, and only slightly reduced the numberof macrophages (21%) to a level that was far less than the 65% reductionobserved following treatment with Formulation VIII at a similar dosingof moles of salt. The p38 MAPK inhibitor ADS110836 reduced cell countsin for each cell type by a statistically significant amount, as wasexpect (Table 73).

TABLE 73 Efficacy of Formulation XXVI and VIII in the TS mouse modelCompound Formulation VIII NaCl Form. XXVI ADS115398 Dose 6 capsules 3capsules 6 capsules 6 capsules 0.1 mg/kg Inflammatory markers in BALInhibition Inhibition Inhibition Inhibition Inhibition % p % p % p % p %p Total cells 53 <0.001 44 <0.001 4 ns 15 ns 48 <0.001 Macrophages 52<0.001 41 <0.001 8 ns 21 <0.05 43 <0.001 Epithelial 37 <0.50 34 ns −3 ns−4 ns 47 <0.01 cells Neutrophils 62 <0.001 54 <0.001 0 ns 13 ns 61<0.001 Eosinophils 84 ns 44 ns −35 ns 52 ns 73 ns Lymphocytes 65 <0.00154 <0.01 −4 ns 20 ns 53 <0.05 ns = not statistically significant

Collectively, the data suggested that calcium-based dry powders had asignificant impact in reducing airway inflammation and are suitabletherapies for treating/preventing neutrophilic inflammation, which isparticularly associated with respiratory diseases like asthma, COPD andCF. Further, that magnesium-based dry powders did not have a significantimpact in reducing airway inflammation and are not suitable therapiesfor treating/preventing neutrophilic inflammation which is particularlyassociated with respiratory diseases like asthma, COPD and CF.

Example 52 Comparison of Calcium and Magnesium Containing Dry Powders toTreat Acute Lipopolysaccharide Inflammation

In this study, a mouse model of acute lung injury was used to study theeffects of calcium and magnesium dry powder formulations on pulmonaryinflammation. Mice were exposed to aerosolized lipopolysaccharide (LPS)isolated from Pseudomonas Aeruginosa. This challenge resulted in lunginflammation and caused changes in pulmonary function. The principlechange in inflammation was an increase in the number of neutrophils inthe lungs and similar changes in lung inflammation and pulmonaryfunction were observed in humans suffering from acute lung injury.

The goal of these studies was to evaluate the efficacy of calciumlactate and magnesium lactate dry powders on pulmonary inflammation. Inthe course of this work it was discovered that both calcium andmagnesium lactate powders significantly reduced pulmonary lunginflammation.

Mice were exposed to whole body exposure with nebulized LPS, 1.12 mg/ml,for 30 minutes. One hour following LPS exposure animals were treatedwith a Placebo-B (98% leucine, 2% NaCl) dry powder, Formulation VIII(20% leucine, 75% calcium lactate, 5% NaCl), or Formulation XXVII (20%leucine, 75% magnesium lactate, 5% NaCl) using a whole body exposurechamber and a capsule based dry powder inhaler system. Animals weretreated with two, 90 mg capsules that would correspond to approximately0.32 mg Ca²⁺/kg delivered to the lung with the calcium lactatecontaining dry powder. Three hours following dry powder treatment allmice were euthanized and underwent whole lung lavage for determinationof total and differential cell counts.

As shown in Table 74, treatment of mice with both Formulations VIII andXXVII significantly reduced total cell counts and neutrophils in the BALfluid when compared with animals exposed to a placebo powder. Thisindicates that both calcium lactate and magnesium lactate dry powdersmay serve as an effective therapy for the treatment of pulmonaryinflammation.

Formulations VIII and XXVII reduce inflammation in a rodent model ofacute lung injury. Placebo-B Formulation VIII Formulation XXVII cells *10⁶/ml Std Dev cells * 10⁶/ml Std Dev cells * 10⁶/ml Std Dev Neutrophils3.39 1.00 1.60 0.52 1.36 0.22 Total cells 3.54 1.05 1.72 0.49 1.55 0.32(Cellularity)

The content of each of the patents, patent applications, patentpublications and published articles cited in this specification areherein incorporated by reference in their entirety.

The invention claimed is:
 1. A respirable dry powder comprisingrespirable dry particles comprising magnesium lactate in an amount thatprovides magnesium ion in an amount of at least about 5%, leucine in anamount of about 5% to about 45%, and one or more therapeutic agents inan amount of about 0.01% to about 20%, wherein all percentages areweight percentages and the magnesium lactate, leucine and one or moretherapeutic agents amount to 100 weight %, wherein the respirable dryparticles have a volume median geometric diameter (VMGD) at 1 bar asmeasured using a HELOS/RODOS system of about 5 microns or less, adispersibility ratio (1/4 bar) of 1.5 or less, and a tap density ofgreater than about 0.4 g/cc.
 2. The respirable dry powder of claim 1,wherein the respirable dry particles have a tap density of about 0.5g/cc to about 1.2 g/cc.
 3. The respirable dry powder of claim 1, whereinsaid magnesium salt does not have a biological activity selected fromthe group consisting of anti-bacterial activity, anti-viral activity,anti-inflammatory activity and combinations thereof.
 4. The respirabledry powder of claim 1, wherein the one or more additional-therapeuticagents are independently selected from the group consisting of LABAs,short-acting beta agonists, corticosteroids, LAMAs, antibiotics, dornasealpha, sodium channel blockers, and combinations thereof.
 5. Therespirable dry powder of claim 1, wherein the respirable dry powder hasa Fine Particle Fraction (FPF) of less than 5.6 microns of at least 45%.6. A method of treating a respiratory disease comprising administeringto the respiratory tract of a patient in need thereof an effectiveamount of a respirable dry powder of claim
 1. 7. A method of treating orpreventing an infectious disease of the respiratory ac comprisingadministering to the respiratory tract of a patient in need thereof aneffective amount of a respirable dry powder of claim
 1. 8. A method ofreducing inflammation comprising administering to the respiratory tractof a patient in need thereof an effective amount of a respirable drypowder of claim 1.